Depleting tumor-specific tregs

ABSTRACT

The present invention provides, among other things, methods and compositions for diagnosing and/or treating cancer by targeting CCR8. In particular, the present invention provides technologies for depleting Treg cells, and particularly tumor-infiltrating Treg cells.

BACKGROUND

Significant effort has been invested in the identification and/ordevelopment for promoting ability of the immune system to target anddestroy tumors. Unfortunately, so far, success has proven elusive.Indeed, although therapeutic modulation of the immune system in cancerpatients through, for example, antibody blockage of inhibitorymolecules, adoptive T cell transfer, vaccination and other methods hasshown some clinical benefit, patient responses have been variable atbest.

SUMMARY

The present invention provides technologies for treating cancer bydepleting Treg cells. In particular, the present invention demonstratesthat targeting CCR8 can achieve specific depletion of Treg cells ascompared with other immune cells including specifically CD4 and/or CD8cells.

Prior to the present disclosure, techniques were not available fortargeting Treg cells without also potentially targeting other immunecells. Given that inadvertent targeting of other immune cells mightinhibit, rather than propose, an anti-tumor immune response, prior tothe present disclosure those skilled in the art might reasonably bediscouraged or dissuaded from attempting therapeutic strategies directedat Treg depletion. The present invention, however, provides mechanismsfor specifically targeting Treg cells, as compared with other immunecells, for depletion. In particular, the present disclosure demonstratesthe feasibility and effectiveness of targeting CCR8 to achieve specificdepletion of Treg cells as compared with CD4 and CD8 cells.

In some embodiments, the present invention provides technologies for thediagnosis and/or treatment of cancer by targeting CCR8, for examplethrough administration of compositions comprising CCR8-targeting agents.

In some embodiments, the present invention provides technologies for theidentification and/or characterization of agents useful in the diagnosisand/or treatment of cancer through detection and/or analysis of agentsthat specifically target CCR8. In some embodiments, the presentinvention provides technologies for the identification and/orcharacterization of agents useful in the diagnosis and/or treatment ofcancer through detection and/or analysis of binding to CCR8. In someembodiments, the present invention provides technologies for theidentification and/or characterization of agents useful in the diagnosisand/or treatment of cancer through detection and/or analysis ofdepletion of tumor-infiltrating Treg cells.

In some embodiments, the present invention provides methods of treatingcancer by targeting CCR8 in a subject having a tumor, so thattumor-infiltrating Treg cells are depleted in the subject. In some suchembodiments, the targeting CCR8 comprises administering to the subject acomposition comprising a CCR8-targeting agent that depletestumor-infiltrating Treg cells. In some embodiments the CCR8-targetingagent binds specifically to CCR8 in or on tumor-infiltrating Treg cells.In some particular embodiments, the CCR8-targeting agent is or comprisesan antibody agent. In some embodiments, the CCR8-targeting agentcomprises a payload moiety so that, when the CCR8-targeting agent isdelivered to tumor-infiltrating Treg cells, such cells are depleted fromthe tumor.

Alternatively or additionally, in some embodiments, CCR8-targetingmethods of the present invention further comprise administering one ormore immunomodulatory therapeutic modalities to the subject.

In some embodiments, the present invention provides methods of detectingand/or characterizing a tumor, and/or Tregs infiltrating it, byadministering to a subject having the tumor a composition comprising aCCR8-targeting agent in association with a detectable moiety, so thattumor-infiltrating Treg cells are detected. In some embodiments, suchmethods further comprise, prior to and/or after the detecting and/orcharacterizing, treating the subject in which tumor-infiltrating Tregcells are detected by targeting CCR8. In some embodiments, providedmethods include selecting subjects and/or tumors for treatment forCCR8-targeted therapy and/or for anti-tumor immunomodulatory therapy.Alternatively or additionally, in some embodiments, provided methodsinclude monitoring progress of cancer and/or of therapy.

Alternatively or additionally, in some embodiments, CCR8-targetingmethods of the present invention further comprise targeting CCR4. Insome such embodiments, targeting CCR4 comprises administering to thesubject a composition comprising a CCR4-targeting agent. In someparticular embodiments, the CCR4-targeting agent is or comprises abifunctional agent that targets both CCR8 and CCR4.

In some embodiments, the present invention provides methods ofidentifying and/or characterizing one or more CCR8-targeting agents bycontacting the one or more agents with CCR8 and determining theirability to bind specifically to CCR8. In some such embodiments, the stepof contacting comprises contacting with isolated CCR8, for example bycontacting with cells expressing CCR8, and/or with tissue is which CCR8is expressed. In some embodiments, the step of contacting comprisesadministering to an organism comprising tissue or cells in which CCR8 isexpressed.

In some embodiments, of provided methods of identifying and/orcharacterizing one or more CCR8-targeting agents by contacting the oneor more agents with CCR8 and determining their ability to bindspecifically to CCR8, the step of determining comprises detectingspecific binding of the one or more agents (e.g., one or more antibodyagents) to tumor-infiltrating Treg cells in the organism.

In some embodiments, the present invention provides methods ofidentifying and/or characterizing one or more CCR8-targeting agents bycontacting the one or more agents with a tumor comprisingtumor-infiltrating Treg cells and determining their ability to depletethe tumor-infiltrating Treg cells from the tumor. In some suchembodiments, the step of contacting comprises administering to anorganism having the tumor.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

Activates: As used herein, the term “activates” refers to increasing thelevel and/or activity of a target.

Activating agent: As used herein, the term “activating agent” refers toan agent whose presence or level correlates with elevated level and/oractivity of a target, as compared with that observed absent the agent(or with the agent at a different level). In some embodiments, anactivating agent is one whose presence or level correlates with a targetlevel or activity that is comparable to or greater than a particularreference level or activity (e.g., that observed under appropriatereference conditions, such as presence of a known activating agent,e.g., a positive control). In some embodiments, an activating agent i)stimulates, promotes, or increases level and/or activity of anotherentity and/or ii) stimulates, promotes, accelerates (e.g., timing and/orfrequency of) or increases one or more effects of such other entity;and/or ii) inhibits, decreases, reduces, or delays one or morebiological events.

Affinity: As is known in the art, “affinity” is a measure of thetightness with a particular ligand binds to its partner. Affinities canbe measured in different ways. In some embodiments, affinity is measuredby a quantitative assay. In some such embodiments, binding partnerconcentration may be fixed to be in excess of ligand concentration so asto mimic physiological conditions. Alternatively or additionally, insome embodiments, binding partner concentration and/or ligandconcentration may be varied. In some such embodiments, affinity may becompared to a reference under comparable conditions (e.g.,concentrations).

Agent: The term “agent” as used herein may refer to a compound or entityof any chemical class including, for example, polypeptides, nucleicacids, saccharides, lipids, small molecules, metals, or combinationsthereof. As will be clear from context, in some embodiments, an agentcan be or comprise a cell or organism, or a fraction, extract, orcomponent thereof. In some embodiments, an agent is agent is orcomprises a natural product in that it is found in and/or is obtainedfrom nature. In some embodiments, an agent is or comprises one or moreentities that is man-made in that it is designed, engineered, and/orproduced through action of the hand of man and/or is not found innature. In some embodiments, an agent may be utilized in isolated orpure form; in some embodiments, an agent may be utilized in crude form.In some embodiments, potential agents are provided as collections orlibraries, for example that may be screened to identify or characterizeactive agents within them. Some particular embodiments of agents thatmay be utilized in accordance with the present invention include smallmolecules, antibodies, antibody fragments, aptamers, siRNAs, shRNAs,DNA/RNA hybrids, antisense oligonucleotides, ribozymes, peptides,peptide mimetics, small molecules, etc. In some embodiments, an agent isor comprises a polymer. In some embodiments, an agent is not a polymerand/or is substantially free of any polymer. In some embodiments, anagent contains at least one polymeric moiety. In some embodiments, anagent lacks or is substantially free of any polymeric moiety.

Analog: As used herein, the term “analog” refers to a substance thatshares one or more particular structural features, elements, components,or moieties with a reference substance. Typically, an “analog” showssignificant structural similarity with the reference substance, forexample sharing a core or consensus structure, but also differs incertain discrete ways. In some embodiments, an analog a substance thatcan be generated from the reference substance by chemical manipulationof the reference substance. In some embodiments, an analog is asubstance that can be generated through performance of a syntheticprocess substantially similar to (e.g., sharing a plurality of stepswith) one that generates the reference substance. In some embodiments,an analog is or can be generated through performance of a syntheticprocess different from that used to generate the reference substance

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to a human, at anappropriate stage of development (including any stage). In someembodiments, “animal” refers to a non-human animals, at an appropriatestage of development (including any stage). In certain embodiments, anon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal is a vertebrate. In some embodiments, an animalmay be a transgenic animal, genetically-engineered animal, and/or aclone.

Antagonist: As used herein, the term “antagonist” refers to an agentwhose presence or level correlates with reduced level or activity of atarget, as compared with that observed absent the agent (or with theagent at a different level). In some embodiments, an antagonist is anagent whose presence or level correlates with a target level or activitythat is comparable to or less than a particular reference level oractivity (e.g., that observed under appropriate reference conditions,such as presence of a known antagonist (e.g., a positive control). Insome embodiments, the term “antagonist” refers to an agent that i)inhibits, decreases or reduces level and/or activity of another entityand/or ii) inhibits, decreases, delays or reduces one or more effects ofsuch other entity; and/or ii) inhibits, decreases, reduces, or delaysone or more biological events. Antagonists may be or include agents ofany chemical class including, for example, small molecules,polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or anyother entity that shows the relevant inhibitory activity. In someembodiments, an antagonist may be direct (in which case it exerts itsinfluence directly upon its target); in some embodiments, an antagonistmay be indirect (in which case it exerts its influence by other thanbinding to its target; e.g., by interacting with a regulator of thetarget, for example so that level or activity of the target is altered).In some embodiments, action of an antagonist may be reversible; in someembodiments it may be irreversible. In some embodiments, an antagonistmay form a covalent bond with its target; in many such embodiments, theantagonist acts as an irreversible inhibitor of that target. In someembodiments, an antagonist interacts with an active site on its target(e.g., a site of interaction with a partner entity or substrate). Insome embodiments, an antagonist competes with another entity (e.g., apartner binding agent or a substrate) for interaction with a target.

Antibody: As used herein, the term “antibody” refers to a polypeptidethat includes canonical immunoglobulin sequence elements sufficient toconfer specific binding to a particular target antigen. As is known inthe art, intact antibodies as produced in nature are approximately 150kD tetrameric agents comprised of two identical heavy chain polypeptides(about 50 kD each) and two identical light chain polypeptides (about 25kD each) that associate with each other into what is commonly referredto as a “Y-shaped” structure. Each heavy chain is comprised of at leastfour domains (each about 110 amino acids long)—an amino-terminalvariable (VH) domain (located at the tips of the Y structure), followedby three constant domains: CH1, CH2, and the carboxy-terminal CH3(located at the base of the Y's stem). A short region, known as the“switch”, connects the heavy chain variable and constant regions. The“hinge” connects CH2 and CH3 domains to the rest of the antibody. Twodisulfide bonds in this hinge region connect the two heavy chainpolypeptides to one another in an intact antibody. Each light chain iscomprised of two domains—an amino-terminal variable (VL) domain,followed by a carboxy-terminal constant (CL) domain, separated from oneanother by another “switch”. Intact antibody tetramers are comprised oftwo heavy chain-light chain dimers in which the heavy and light chainsare linked to one another by a single disulfide bond; two otherdisulfide bonds connect the heavy chain hinge regions to one another, sothat the dimers are connected to one another and the tetramer is formed.Naturally-produced antibodies are also glycosylated, typically on theCH2 domain. Each domain in a natural antibody has a structurecharacterized by an “immunoglobulin fold” formed from two beta sheets(e.g., 3-, 4-, or 5-stranded sheets) packed against each other in acompressed antiparallel beta barrel. Each variable domain contains threehypervariable loops known as “complement determining regions” (CDR1,CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1,FR2, FR3, and FR4). When natural antibodies fold, the FR regions formthe beta sheets that provide the structural framework for the domains,and the CDR loop regions from both the heavy and light chains arebrought together in three-dimensional space so that they create a singlehypervariable antigen binding site located at the tip of the Ystructure. Amino acid sequence comparisons among antibody polypeptidechains have defined two light chain (κ and λ) classes, several heavychain (e.g., μ, γ, α, ε, δ) classes, and certain heavy chain subclasses(α1, α2, γ1, γ2, γ3, and γ4). Antibody classes (IgA [including IgA1,IgA2], IgD, IgE, IgG [including IgG1, IgG2, IgG3, IgG4], IgM) aredefined based on the class of the utilized heavy chain sequences. The Fcregion of naturally-occurring antibodies binds to elements of thecomplement system, and also to receptors on effector cells, includingfor example effector cells that mediate cytotoxicity. As is known in theart, affinity and/or other binding attributes of Fc regions for Fcreceptors can be modulated through glycosylation or other modification.In some embodiments, antibodies produced and/or utilized in accordancewith the present invention include glycosylated Fc domains, including Fcdomains with modified or engineered such glycosylation For purposes ofthe present invention, in certain embodiments, any polypeptide orcomplex of polypeptides that includes sufficient immunoglobulin domainsequences as found in natural antibodies can be referred to and/or usedas an “antibody”, whether such polypeptide is naturally produced (e.g.,generated by an organism reacting to an antigen), or produced byrecombinant engineering, chemical synthesis, or other artificial systemor methodology. In some embodiments, an antibody is polyclonal; in someembodiments, an antibody is monoclonal. In some embodiments, an antibodyhas constant region sequences that are characteristic of mouse, rabbit,primate, or human antibodies. In some embodiments, antibody sequenceelements are humanized, primatized, chimeric, etc, as is known in theart. Moreover, the term “antibody” as used herein, will be understood toencompass (unless otherwise stated or clear from context) can refer inappropriate embodiments to any of the art-known or developed constructsor formats for capturing antibody structural and functional features inalternative presentation. For example, in some embodiments, the term canrefer to bi- or other multi-specific (e.g., zybodies, etc) antibodies,Small Modular ImmunoPharmaceuticals (“SMIPs™”), single chain antibodies,cameloid antibodies, and/or antibody fragments. In some embodiments, anantibody may lack a covalent modification (e.g., attachment of a glycan)that it would have if produced naturally. In some embodiments, anantibody may contain a covalent modification (e.g., attachment of aglycan, a payload [e.g., a detectable moiety, a therapeutic moiety, acatalytic moiety, etc], or other pendant group [e.g., poly-ethyleneglycol, etc.

Antibody agent: As used herein, the term “antibody agent” refers to anagent that specifically binds to a particular antigen. In someembodiments, the term encompasses any polypeptide with immunoglobulinstructural elements sufficient to confer specific binding. Suitableantibody agents include, but are not limited to, human antibodies,primatized antibodies, chimeric antibodies, bi-specific antibodies,humanized antibodies, conjugated antibodies (i.e., antibodies conjugatedor fused to other proteins, radiolabels, cytotoxins), Small ModularImmunoPharmaceuticals (“SMIPs™”), single chain antibodies, cameloidantibodies, and antibody fragments. As used herein, the term “antibodyagent” also includes intact monoclonal antibodies, polyclonalantibodies, single domain antibodies (e.g., shark single domainantibodies (e.g., IgNAR or fragments thereof)), multispecific antibodies(e.g. bi-specific antibodies) formed from at least two intactantibodies, and antibody fragments so long as they exhibit the desiredbiological activity. In some embodiments, the term encompasses stapledpeptides. In some embodiments, the term encompasses one or moreantibody-like binding peptidomimetics. In some embodiments, the termencompasses one or more antibody-like binding scaffold proteins. In comeembodiments, the term encompasses monobodies or adnectins. In manyembodiments, an antibody agent is or comprises a polypeptide whose aminoacid sequence includes one or more structural elements recognized bythose skilled in the art as a complementarity determining region (CDR);in some embodiments an antibody agent is or comprises a polypeptidewhose amino acid sequence includes at least one CDR (e.g., at least oneheavy chain CDR and/or at least one light chain CDR) that issubstantially identical to one found in a reference antibody. In someembodiments an included CDR is substantially identical to a referenceCDR in that it is either identical in sequence or contains between 1-5amino acid substitutions as compared with the reference CDR. In someembodiments an included CDR is substantially identical to a referenceCDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with thereference CDR. In some embodiments an included CDR is substantiallyidentical to a reference CDR in that it shows at least 96%, 96%, 97%,98%, 99%, or 100% sequence identity with the reference CDR. In someembodiments an included CDR is substantially identical to a referenceCDR in that at least one amino acid within the included CDR is deleted,added, or substituted as compared with the reference CDR but theincluded CDR has an amino acid sequence that is otherwise identical withthat of the reference CDR. In some embodiments an included CDR issubstantially identical to a reference CDR in that 1-5 amino acidswithin the included CDR are deleted, added, or substituted as comparedwith the reference CDR but the included CDR has an amino acid sequencethat is otherwise identical to the reference CDR . In some embodimentsan included CDR is substantially identical to a reference CDR in that atleast one amino acid within the included CDR is substituted as comparedwith the reference CDR but the included CDR has an amino acid sequencethat is otherwise identical with that of the reference CDR. In someembodiments an included CDR is substantially identical to a referenceCDR in that 1-5 amino acids within the included CDR are deleted, added,or substituted as compared with the reference CDR but the included CDRhas an amino acid sequence that is otherwise identical to the referenceCDR. In some embodiments, an antibody agent is or comprises apolypeptide whose amino acid sequence includes structural elementsrecognized by those skilled in the art as an immunoglobulin variabledomain. In some embodiments, an antibody agent is a polypeptide proteinhaving a binding domain which is homologous or largely homologous to animmunoglobulin-binding domain.

Antibody fragment: As used herein, an “antibody fragment” includes aportion of an intact antibody, such as, for example, the antigen-bindingor variable region of an antibody. Examples of antibody fragmentsinclude Fab, Fab′, F(ab′)2, and Fv fragments; triabodies; tetrabodies;linear antibodies; single-chain antibody molecules; and multi specificantibodies formed from antibody fragments. For example, antibodyfragments include isolated fragments, “Fv” fragments, consisting of thevariable regions of the heavy and light chains, recombinant single chainpolypeptide molecules in which light and heavy chain variable regionsare connected by a peptide linker (“ScFv proteins”), and minimalrecognition units consisting of the amino acid residues that mimic thehypervariable region. In many embodiments, an antibody fragment containssufficient sequence of the parent antibody of which it is a fragmentthat it binds to the same antigen as does the parent antibody; in someembodiments, a fragment binds to the antigen with a comparable affinityto that of the parent antibody and/or competes with the parent antibodyfor binding to the antigen. Examples of antigen binding fragments of anantibody include, but are not limited to, Fab fragment, Fab′ fragment,F(ab′)2 fragment, scFv fragment, Fv fragment, dsFv diabody, dAbfragment, Fd′ fragment, Fd fragment, and an isolated complementaritydetermining region (CDR) region. An antigen binding fragment of anantibody may be produced by any means. For example, an antigen bindingfragment of an antibody may be enzymatically or chemically produced byfragmentation of an intact antibody and/or it may be recombinantlyproduced from a gene encoding the partial antibody sequence.Alternatively or additionally, antigen binding fragment of an antibodymay be wholly or partially synthetically produced. An antigen bindingfragment of an antibody may optionally comprise a single chain antibodyfragment. Alternatively or additionally, an antigen binding fragment ofan antibody may comprise multiple chains which are linked together, forexample, by disulfide linkages. An antigen binding fragment of anantibody may optionally comprise a multimolecular complex. A functionalantibody fragment typically comprises at least about 50 amino acids andmore typically comprises at least about 200 amino acids.

Antigen: As used herein, the term “antigen” refers to a molecule orentity to which an immunoglobulin specifically binds. In someembodiments, an antigen is or comprises a polypeptide or portionthereof. In some embodiments, an antigen is an agent that elicits animmune response; and/or (ii) an agent that is bound by a T cell receptor(e.g., when presented by an WIC molecule) or to an antibody (e.g.,produced by a B cell) when exposed or administered to an organism. Insome embodiments, an antigen elicits a humoral response (e.g., includingproduction of antigen-specific antibodies) in an organism; alternativelyor additionally, in some embodiments, an antigen elicits a cellularresponse (e.g., involving T-cells whose receptors specifically interactwith the antigen) in an organism. It will be appreciated by thoseskilled in the art that a particular antigen may elicit an immuneresponse in one or several members of a target organism (e.g., mice,rabbits, primates, humans), but not in all members of the targetorganism species. In some embodiments, an antigen elicits an immuneresponse in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ofthe members of a target organism species. In some embodiments, anantigen binds to an antibody and/or T cell receptor, and may or may notinduce a particular physiological response in an organism. In someembodiments, for example, an antigen may bind to an antibody and/or to aT cell receptor in vitro, whether or not such an interaction occurs invivo. In general, an antigen may be or include any chemical entity suchas, for example, a small molecule, a nucleic acid, a polypeptide, acarbohydrate, a lipid, a polymer [in some embodiments other than abiologic polymer (e.g., other than a nucleic acid or amino acidpolymer)] etc. In some embodiments, an antigen is or comprises apolypeptide. In some embodiments, an antigen is or comprises a glycan.Those of ordinary skill in the art will appreciate that, in general, anantigen may be provided in isolated or pure form, or alternatively maybe provided in crude form (e.g., together with other materials, forexample in an extract such as a cellular extract or other relativelycrude preparation of an antigen-containing source). In some embodiments,antigens utilized in accordance with the present invention are providedin a crude form. In some embodiments, an antigen is or comprises arecombinant antigen.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Binding: As used herein, the term “binding” refers to a non-covalentassociation between or among two or more entities. “Direct” bindinginvolves physical contact between entities or moieties; indirect bindinginvolves physical interaction by way of physical contact with one ormore intermediate entities. Binding between two or more entities can beassessed in any of a variety of contexts—including where interactingentities or moieties are studied in isolation or in the context of morecomplex systems (e.g., while covalently or otherwise associated with acarrier entity and/or in a biological system or cell).

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active. In particularembodiments, where a peptide is biologically active, a portion of thatpeptide that shares at least one biological activity of the peptide istypically referred to as a “biologically active” portion. In certainembodiments, a peptide has no intrinsic biological activity but thatinhibits the effects of one or more naturally-occurring angiotensincompounds is considered to be biologically active.

Carrier or diluent: As used herein, the terms “carrier” and “diluent”refers to a pharmaceutically acceptable (e.g., safe and non-toxic foradministration to a human) carrier or diluting substance useful for thepreparation of a pharmaceutical formulation. Exemplary diluents includesterile water, bacteriostatic water for injection (BWFI), a pH bufferedsolution (e.g., phosphate-buffered saline), sterile saline solution,Ringer's solution or dextrose solution.

Characteristic portion: As used herein, the term a “characteristicportion” of a substance, in the broadest sense, is one that shares somedegree of sequence or structural identity with respect to the wholesubstance. In certain embodiments, a characteristic portion shares atleast one functional characteristic with the intact substance. Forexample, a “characteristic portion” of a protein or polypeptide is onethat contains a continuous stretch of amino acids, or a collection ofcontinuous stretches of amino acids, that together are characteristic ofa protein or polypeptide. In some embodiments, each such continuousstretch generally contains at least 2, 5, 10, 15, 20, 50, or more aminoacids. In general, a characteristic portion of a substance (e.g., of aprotein, antibody, etc.) is one that, in addition to the sequence and/orstructural identity specified above, shares at least one functionalcharacteristic with the relevant intact substance; epitope-bindingspecificity is one example. In some embodiments, a characteristicportion may be biologically active.

Combination therapy: As used herein, the term “combination therapy”refers to those situations in which two or more different pharmaceuticalagents for the treatment of disease are administered in overlappingregimens so that the subject is simultaneously exposed to at least twoagents. In some embodiments, the different agents are administeredsimultaneously. In some embodiments, the administration of one agentoverlaps the administration of at least one other agent. In someembodiments, the different agents are administered sequentially suchthat the agents have simultaneous biologically activity with in asubject.

Comparable: The term “comparable” is used herein to describe two (ormore) sets of conditions, circumstances, individuals, or populationsthat are sufficiently similar to one another to permit comparison ofresults obtained or phenomena observed. In some embodiments, comparablesets of conditions, circumstances, individuals, or populations arecharacterized by a plurality of substantially identical features and oneor a small number of varied features. Those of ordinary skill in the artwill appreciate that sets of circumstances, individuals, or populationsare comparable to one another when characterized by a sufficient numberand type of substantially identical features to warrant a reasonableconclusion that differences in results obtained or phenomena observedunder or with different sets of circumstances, individuals, orpopulations are caused by or indicative of the variation in thosefeatures that are varied.

Dendritic cell: As used herein, the term “dendritic cell” refers toparticular immune cells that process antigen material and present it onthe surface to other cells of the immune system. Dendritic cells act asmessengers between the innate and adaptive immunity and arecommunicating with other cells through direct contact or at a distanceusing cytokines. Reacting to the presence of foreign antigens, dendriticcells produce cytokines which in turn induce other immune cells, T cellsfor example, to aid in the immune response.

Diagnostic information: As used herein, the term “diagnosticinformation” or information for use in diagnosis is any information thatis useful in determining whether a patient has a disease or conditionand/or in classifying the disease or condition into a phenotypiccategory or any category having significance with regard to prognosis ofthe disease or condition, or likely response to treatment (eithertreatment in general or any particular treatment) of the disease orcondition. Similarly, diagnosis refers to providing any type ofdiagnostic information, including, but not limited to, whether a subjectis likely to have a disease or condition (such as cancer), state,staging or characteristic of the disease or condition as manifested inthe subject, information related to the nature or classification of atumor, information related to prognosis and/or information useful inselecting an appropriate treatment. Selection of treatment may includethe choice of a particular therapeutic (e.g., chemotherapeutic) agent orother treatment modality such as surgery, radiation, etc., a choiceabout whether to withhold or deliver therapy, a choice relating todosing regimen (e.g., frequency or level of one or more doses of aparticular therapeutic agent or combination of therapeutic agents), etc.

Dosage form: As used herein, the terms “dosage form” and “unit dosageform” refer to a physically discrete unit of a therapeutic agent for thepatient to be treated. Each unit contains a predetermined quantity ofactive material calculated to produce the desired therapeutic effect. Itwill be understood, however, that the total dosage of the compositionwill be decided by the attending physician within the scope of soundmedical judgment.

Dosing regimen: A “dosing regimen” (or “therapeutic regimen”), as thatterm is used herein, is a set of unit doses (typically more than one)that are administered individually to a subject, typically separated byperiods of time. In some embodiments, a given therapeutic agent has arecommended dosing regimen, which may involve one or more doses. In someembodiments, a dosing regimen comprises a plurality of doses each ofwhich are separated from one another by a time period of the samelength; in some embodiments, a dosing regimen comprises a plurality ofdoses and at least two different time periods separating individualdoses. In some embodiments, the therapeutic agent is administeredcontinuously over a predetermined period. In some embodiments, thetherapeutic agent is administered once a day (QD) or twice a day (BID).

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end formation); (3) translation of an RNA into a polypeptide orprotein; and/or (4) post-translational modification of a polypeptide orprotein.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized. A biological molecule may havetwo functions (i.e., bifunctional) or many functions (i.e.,multifunctional).

Gene: As used herein, the term “gene” has its meaning as understood inthe art. In some embodiments, the term “gene” may include generegulatory sequences (e.g., promoters, enhancers, etc.) and/or intronsequences. In some embodiments, the term refers to nucleic acids that donot encode proteins but rather encode functional RNA molecules such astRNAs, RNAi-inducing agents, etc. Alternatively or additionally, in manyembodiments, the term “gene”, as used in the present application, refersto a portion of a nucleic acid that encodes a protein. Whether the termencompasses other sequences (e.g., non-coding sequences, regulatorysequences, etc) will be clear from context to those of ordinary skill inthe art.

Gene product or expression product: As used herein, the term “geneproduct” or “expression product” generally refers to an RNA transcribedfrom the gene (pre-and/or post-processing) or a polypeptide (pre- and/orpost-modification) encoded by an RNA transcribed from the gene.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to an appropriate baseline or reference level or amount.Those of ordinary skill in the art will be aware of appropriatereference levels or amount for particular values of interest inaccordance with the present invention. To give but a few examples, insome embodiments, a reference level or amount is that determined underotherwise comparable conditions (e.g., in the same system or individual)absent administration of a particular agent. In some embodiments areference level or amount is that determined in an appropriatecomparator system, individual, or population (e.g., in a system,individual or population not afflicted with or representative of aparticular disease, disorder or condition).

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism.

In vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism, such as a human and a non-humananimal. In the context of cell-based systems, the term may be used torefer to events that occur within a living cell (as opposed to, forexample, in vitro systems).

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of theother components with which they were initially associated. In someembodiments, isolated agents are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, substantially 100%, or 100% pure. Asused herein, a substance is “pure” if it is substantially free of othercomponents. As used herein, the term “isolated cell” refers to a cellthat is not presently part of a multi-cellular organism.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition is or may be administered,e.g., for experimental, diagnostic, prophylactic, cosmetic, and/ortherapeutic purposes. Typical patients include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and/or humans). In someembodiments, a patient is a human. In some embodiments, a patient issuffering from or susceptible to one or more disorders or conditions. Insome embodiments, a patient displays one or more symptoms of a disorderor condition. In some embodiments, a patient has been diagnosed with oneor more disorders or conditions. In some embodiments, the disorder orcondition is or includes inflammation.

Pharmaceutically acceptable: As used herein, the term “pharmaceuticallyacceptable”, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Pharmaceutical composition: As used herein, the term “pharmaceuticalcomposition” refers to an active agent, formulated together with one ormore pharmaceutically acceptable carriers. In some embodiments, activeagent is present in unit dose amount appropriate for administration in atherapeutic regimen that shows a statistically significant probabilityof achieving a predetermined therapeutic effect when administered to arelevant population. In some embodiments, pharmaceutical compositionsmay be specially formulated for administration in solid or liquid form,including those adapted for the following: oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation;topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream, or foam; sublingually; ocularly; transdermally; or nasally,pulmonary, and to other mucosal surfaces.

Pharmaceutically acceptable: The term “pharmaceutically acceptable” asused herein, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Prevent: As used herein, the term “prevent” or “prevention”, when usedin connection with the occurrence of a disease, disorder, and/orcondition, refers to reducing the risk of developing the disease,disorder and/or condition and/or to delaying the onset of one or moresymptoms or characteristics of the disease, disorder, or condition. Seethe definition of “risk.”

Prognostic and predictive information: As used herein, the terms“prognostic and predictive information” are used interchangeably torefer to any information that may be used to indicate any aspect of thecourse of a disease or condition either in the absence or presence oftreatment. Such information may include, but is not limited to, theaverage life expectancy of a patient, the likelihood that a patient willsurvive for a given amount of time (e.g., 6 months, 1 year, 5 years,etc.), the likelihood that a patient will be cured of a disease, thelikelihood that a patient's disease will respond to a particular therapy(wherein response may be defined in any of a variety of ways).Prognostic and predictive information are included within the broadcategory of diagnostic information.

Protein: The term “protein” as used herein refers to one or morepolypeptides that function as a discrete unit. If a single polypeptideis the discrete functioning unit and does not require permanent ortemporary physical association with other polypeptides in order to formthe discrete functioning unit, the terms “polypeptide” and “protein” maybe used interchangeably. If the discrete functional unit is comprised ofmore than one polypeptide that physically associate with one another,the term “protein” refers to the multiple polypeptides that arephysically coupled and function together as the discrete unit.

Reference: The term “reference” is often used herein to describe astandard or control agent, individual, population, sample, sequence orvalue against which an agent, individual, population, sample, sequenceor value of interest is compared. In some embodiments, a referenceagent, individual, population, sample, sequence or value is testedand/or determined substantially simultaneously with the testing ordetermination of the agent, individual, population, sample, sequence orvalue of interest. In some embodiments, a reference agent, individual,population, sample, sequence or value is a historical reference,optionally embodied in a tangible medium. Typically, as would beunderstood by those skilled in the art, a reference agent, individual,population, sample, sequence or value is determined or characterizedunder conditions comparable to those utilized to determine orcharacterize the agent, individual, population, sample, sequence orvalue of interest

Response: As used herein, a “response” to treatment may refer to anyalteration in a subject's condition that occurs as a result of orcorrelates with treatment. Such alteration may include a beneficialalteration, such as stabilization of the condition (e.g., prevention ofdeterioration that would have taken place in the absence of thetreatment), amelioration of symptoms of the condition, and/orimprovement in the prospects for cure of the condition, etc. It mayrefer to a subject's response. Subject response may be measuredaccording to a wide variety of criteria, including clinical criteria andobjective criteria. In some embodiments, a response may include analteration that is not beneficial (e.g., a side effect).

Risk: As will be understood from context, a “risk” of a disease,disorder, and/or condition comprises a likelihood that a particularindividual will develop a disease, disorder, and/or condition (e.g.,cancer). In some embodiments, risk is expressed as a percentage. In someembodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed asa risk relative to a risk associated with a reference sample or group ofreference samples. In some embodiments, a reference sample or group ofreference samples have a known risk of a disease, disorder, conditionand/or event (e.g., cancer). In some embodiments a reference sample orgroup of reference samples are from individuals comparable to aparticular individual. In some embodiments, relative risk is 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more.

Sample: As used herein, a sample obtained from a subject may include,but is not limited to, one or more of the following: a cell or cells, aportion of tissue, blood, serum, ascites, urine, saliva, and other bodyfluids, secretions, or excretions. The term “sample” also includes anymaterial derived by processing such a sample. Derived samples mayinclude nucleotide molecules or polypeptides extracted from the sampleor obtained by subjecting the sample to techniques such as amplificationor reverse transcription of mRNA, etc.

Specific binding: As used herein, the terms “specific binding” or“specific for” or “specific to” refer to an interaction (typicallynon-covalent) between a target entity (e.g., a target protein orpolypeptide) and a binding agent (e.g., an antibody, such as a providedantibody). As will be understood by those of ordinary skill, aninteraction is considered to be “specific” if it is favored in thepresence of alternative interactions. In many embodiments, aninteraction is typically dependent upon the presence of a particularstructural feature of the target molecule such as an antigenicdeterminant or epitope recognized by the binding molecule. For example,if an antibody is specific for epitope A, the presence of a polypeptidecontaining epitope A or the presence of free unlabeled A in a reactioncontaining both free labeled A and the antibody thereto, will reduce theamount of labeled A that binds to the antibody. It is to be understoodthat specificity need not be absolute. For example, it is well known inthe art that numerous antibodies cross-react with other epitopes inaddition to those present in the target molecule. Such cross-reactivitymay be acceptable depending upon the application for which the antibodyis to be used. Specificity may be evaluated in the context of additionalfactors such as the affinity of the binding molecule for the targetmolecule versus the affinity of the binding molecule for other targets(e.g., competitors). If a binding molecule exhibits a high affinity fora target molecule that it is desired to detect and low affinity fornon-target molecules, the antibody will likely be an acceptable reagentfor immunodiagnostic purposes. Once the specificity of a bindingmolecule is established in one or more contexts, it may be employed inother, preferably similar, contexts without necessarily re-evaluatingits specificity.

Subject: As used herein, the term “subject” refers to a human or anynon-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate). A human includes pre- and post-natal forms. Inmany embodiments, a subject is a human being. A subject can be apatient, which refers to a human presenting to a medical provider fordiagnosis or treatment of a disease. The term “subject” is used hereininterchangeably with “individual” or “patient.” A subject can beafflicted with or is susceptible to a disease or disorder but may or maynot display symptoms of the disease or disorder.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of the disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition may not exhibitsymptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,condition, or event (for example, cancer) may be characterized by one ormore of the following: (1) a genetic mutation associated withdevelopment of the disease, disorder, and/or condition; (2) a geneticpolymorphism associated with development of the disease, disorder,and/or condition; (3) increased and/or decreased expression and/oractivity of a protein associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, condition, and/or event. In some embodiments, anindividual who is susceptible to a disease, disorder, and/or conditionwill develop the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition will not develop the disease, disorder, and/orcondition.

Symptoms are reduced: According to the present invention, “symptoms arereduced” when one or more symptoms of a particular disease, disorder orcondition is reduced in magnitude (e.g., intensity, severity, etc.)and/or frequency. For purposes of clarity, a delay in the onset of aparticular symptom is considered one form of reducing the frequency ofthat symptom. Many cancer patients with smaller tumors have no symptoms.It is not intended that the present invention be limited only to caseswhere the symptoms are eliminated. The present invention specificallycontemplates treatment such that one or more symptoms is/are reduced(and the condition of the subject is thereby “improved”), albeit notcompletely eliminated.

T cell: As used herein, the term “T cell” refers to a lymphocyte (e.g.,white blood cell) that functions in cell-mediated immunity. In someembodiments, the presence of a T cell receptor (TCR) on the cell surfacedistinguishes T cells from other lymphocytes. As is known in the art, Tcells typically do not present antigens, and rely on other lymphocytes(e.g., natural killer cells and B cells) to aid in antigen presentation.Types of T cells include: T helper cells (TH cells), Memory T cells(Tcm, Tem, or Temra), Regulatory T cells (Treg), Cytotoxic T cells(CTLs), Natural killer T cells (NK cells), gamma delta T cells, andMucosal associated invariant T cells (MAIT).

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that has a therapeutic effect and/or elicits a desiredbiological and/or pharmacological effect, when administered to asubject.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” refers to an amount of a therapeuticagent (e.g., bacterial metabolites, short chain fatty acids, HDACinhibitors) which confers a therapeutic effect on the treated subject,at a reasonable benefit/risk ratio applicable to any medical treatment.The therapeutic effect may be objective (i.e., measurable by some testor marker) or subjective (i.e., subject gives an indication of or feelsan effect). In particular, the “therapeutically effective amount” refersto an amount of a therapeutic protein or composition effective to treat,ameliorate, or prevent a desired disease or condition, or to exhibit adetectable therapeutic or preventative effect, such as by amelioratingsymptoms associated with the disease, preventing or delaying the onsetof the disease, and/or also lessening the severity or frequency ofsymptoms of the disease. A therapeutically effective amount is commonlyadministered in a dosing regimen that may comprise multiple unit doses.For any particular therapeutic protein, a therapeutically effectiveamount (and/or an appropriate unit dose within an effective dosingregimen) may vary, for example, depending on route of administration, oncombination with other pharmaceutical agents. Also, the specifictherapeutically effective amount (and/or unit dose) for any particularpatient may depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the activity of thespecific pharmaceutical agent employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and/orrate of excretion or metabolism of the specific fusion protein employed;the duration of the treatment; and like factors as is well known in themedical arts.

Treating: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof and/or reduce incidence of one or more symptoms or features of aparticular disease, disorder, and/or condition. Treatment may beadministered to a subject who does not exhibit signs of a disease and/orexhibits only early signs of the disease for the purpose of decreasingthe risk of developing pathology associated with the disease.Alternatively or additionally, such treatment may be of a subject whoexhibits one or more established signs of the relevant disease, disorderand/or condition. In some embodiments, treatment may be of a subject whohas been diagnosed as suffering from the relevant disease, disorder,and/or condition. In some embodiments, treatment may be of a subjectknown to have one or more susceptibility factors that are statisticallycorrelated with increased risk of development of the relevant disease,disorder, and/or condition.

Treg cells: As used herein, the term Treg cell refers to Regulatory Tcells (Treg), also sometimes referred to as Suppressor T cells. Tregcells maintain immunological tolerance. During an immune response, Tregsstop T cell-mediated immunity and suppress auto-reactive T cells thathave escaped negative selection within the thymus. Treg cells have alsobeen described as able to suppress other types of immune cells such asNK cells and B cells. Adaptive Treg cells (called Th3 or Tr1 cells) arethought to be generated during an immune response. Naturally occurringTreg cells (CD4+CD25+FoxP3+ Treg cells) are generated in the thymus andhave been linked to interactions between developing T cells with bothmyeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that havebeen activated with the cytokine thymic stromal lymphopoietin (TSLP).The presence of FoxP3 in naturally occurring Treg cells distinguishesthem from other T cells. Mutations of the FOXP3 gene can preventregulatory T cell development, causing the fatal autoimmune diseaseIPEX. In some embodiments, Treg cells and/or populations thereof aredefined by presence of particular surface markers. In some embodiments,human Treg cells and/or populations thereof, are defined byCD3+CD4+CD45RA-CD45RO+CD25+ (>95% Foxp3+, FIG. 4).

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1, Panels A-C: FIG. 1, comprised of Panels A-C, illustrates howAblation of Treg cells affects the growth of fully established primaryand lung metastatic tumors. (A) Flow cytometric quantification ofintratumoral CD4⁺ Foxp3⁺ T cells (left panel) and IFN-γ production in Tcells (right panels). Top: control, Bottom: DT-treated. (B) Growthkinetics of orthotopic tumors in mice treated with 50 μg/kg DT whentumors reached approximately 250 mm³. Representative of two independentexperiments; n=5 mice per group; p<0.0001**** (C) Fraction andrepresentative image of mice with detectable lung metastasis uponbioluminescence imaging of the dissected lungs from the group depictedin (B).

FIG. 2, Panels A-B: FIG. 1, comprised of Panels A-B, depicts levels ofTreg cells present in various human cancers (specifically, breastcarcinoma, melanoma, and colon carcinoma), and of CD4+ tumorinfiltrating lymphocytes in human breast carcinoma. (A) Gates indicateCD4⁺Foxp3⁺ Treg. Ratio of CD8 to CD4 T cell yields. (B) Thirty-fivehuman breast tumor samples were processed and CD4 and CD8 T cell subsetsisolated by fluorescence activated cell sorting. Graph indicates theratio of CD8 to CD4 T cell yields.

FIG. 3, Panels A-C: FIG. 3, comprised of Panels A-C, depicts selectiveinfiltration of breast cancers by Treg cells. CD3⁺ T cells from normalbreast parenchyma and tumor infiltrating lymphocytes from human breastcarcinoma (A). Gates indicate % of CD4⁺Foxp3⁺ Treg. Lymphocytes isolatedfrom normal breast parenchyma and breast cancer from the same patientwere stimulated in vitro with PMA/Ionomycin and analyzed forintracellular cytokine production of IFNγ (B) and TNFα (C). Gatesindicate % of cytokine producing CD3+ T cells.

FIG. 4: FIG. 4 shows fluorescence activated cell sorting strategy forisolation of Treg from human breast tumors. Treg (defined asCD3⁺CD4⁺CD45RA⁻ CD45RO⁺CD25⁺) are 95% Foxp3⁺ following intracellularstaining and flow cytometry analysis with a Foxp3-specific antibody.Foxp3 is a Treg specific transcription factor.

FIG. 5: FIG. 5 depicts levels of CCR8 mRNA transcripts in FACS-isolatedtumor-infiltrating CD8 T cells (tCD8), CD4 T cells (tCD4), Treg cells(tTreg), as well as in normal blood CD4 T cells (bCD4), and Treg cells(bTreg).

FIG. 6: FIG. 6 shows CCR8 protein expression by flow cytometric analysisof human T cells from an invasive breast ductal carcinoma, normalglandular breast tissue, and an adenocarcinoma of the colon.

FIG. 7: FIG. 7 shows CCR8 and CCR4 protein expression by flow cytometricanalysis of human T cells infiltrating an invasive breast ductalcarcinoma.

FIG. 8: FIG. 8 shows a schematic of a mixed bone marrow chimeraexperiment. This experimental strategy takes advantage of the fact thatT cell deficient mice reconstituted with bone marrow from FOXP3^(−/−)mice do not have any Treg cells as FOXP3 is the master transcriptionalregulator of Treg cell development and function. Tcr beta deltadeficient (βδ−/−) mice were sublethally irradiated and reconstitutedwith FOXP3−/− bone marrow and an equal amount of either wild type (WT)or CCR8^(−/−) bone marrow. The resulting mice are mixed bone marrowchimeras with Group 1 only having WT Treg cells and Group 2 only havingCCR8^(−/−) Treg cells. The remaining immune cells are a mixture of thedonor bone marrows.

FIG. 9, Panels A-B: FIG. 9 shows CCR8 expressing Treg cells promoteprimary and metastatic mammary tumor progression. A mammary carcinomacell line generated from C57BL/6 mice expressing a transgene encodingthe PyMT oncogene under control of the MMTV promoter experimental groupswere implanted in the mammary fat pads of mice from the experimentalgroups outlined in FIG. 1. Primary tumor growth was assessed by serialvolume measurements and metastatic lung burden was assessed by countinglung tumors from sacrificed animals under a dissecting microscope. A.Mice lacking CCR8 expressing Treg cells exhibit delayed primary tumorgrowth as compared to the control group. B. The lack of CCR8 expressionon Treg cells also leads to a significant reduction in metastatic lungburden.

FIG. 10, Panels A-B: FIG. 10 shows CCR8 expression on human tumorinfiltrating Treg cells correlates with Treg cell proliferation andhigher tumor grade. Tumor infiltrating Treg cell proliferative status(Ki67 expression) and CCR8 expression was determined by flow cytometryof tumor infiltrating lymphocytes from fresh primary human breastcarcinomas. A. Treg cell Ki67 expression positively correlates with Tregcell CCR8 expression. Ki67 expression is indicated as the % of Tregcells expressing the proliferation marker Ki67. CCR8 expression of Tregcells is indicated as a ratio of the mean fluorescence intensity (MFI)of CCR8 staining divided by that of effector CD4 T cells. B. CCR8expression on tumor infiltrating Treg cells is associated with highertumor grade.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention demonstrates, among other things, that CCR8 ishighly and specifically expressed by tumor-infiltrating Treg cells (ascompared, for example, to tumor-infiltrating effector T cells), andfurthermore demonstrates the generality of this phenomenon across avariety of tumor types. The present invention provides methods andcompositions for the treatment of cancer by targeting CCR8, and/or foridentifying and/or characterizing useful cancer therapeutic and/ordiagnostic agents that target CCR8.

Tress and Immune Evasion

The solid tumor microenvironment contains a variety of immune cells.Extensive human and mouse experimental studies suggest that the typesand properties of the immune cells residing within a tumor influenceclinical response (Refs: 11 and 12, and 22-30). In particular,regulatory T (Treg) cell presence is associated with poor clinicaloutcome in melanoma (Ref: 22), breast (Refs: 23-24) gastric (Ref: 25),ovarian (Ref: 26), pancreatic (Ref: 27) and other cancer types, while ahigh CD8+ tumor infiltrating lymphocyte (TIL) density correlates withimproved survival in several cancer types (Refs: 12, and 28-30). Inlarge cohorts of human colorectal tumors, immunological parameters(type, density, location of immune cells within a tumor) were a betterpredictor of survival than the current histopathological methods usedfor staging (Ref: 11).

Regulatory T cells (Treg) are a subset of CD4 T cells that are requiredfor control of autoimmunity, dampening excessive inflammation caused bythe immune response to pathogens, and maintaining maternal-fetaltolerance (Refs 6-8). Regulatory T cells (Treg) are important inmaintaining homeostasis, controlling the magnitude and duration of theinflammatory response, and in preventing autoimmune and allergicresponses. There are two major classifications of Treg: natural Treg andperipheral Treg. Natural Treg, (nTreg) are a class of thymicallygenerated T-cells while peripheral Treg (pTreg) develop in the peripheryfrom naïve T cells in response to signals such as low doses of antigen,presence of certain microbes, lymphopenia or, in some cases, throughactivation by immature dendritic cells. In some cases, pTreg are thoughtto be generated in response to inflammatory conditions, particularlythose which may be due at least in part to the absence of nTreg cells.

The Forkhead box P3 transcription factor (Foxp3) has been shown to be akey regulator in the differentiation and activity of Treg. In fact,loss-of-function mutations in the Foxp3 gene have been shown to lead tothe lethal IPEX syndrome (immune dysregulation, polyendocrinopathy,enteropathy, X-linked). Patients with IPEX suffer from severe autoimmuneresponses, persistent eczema, and colitis.

In general Tregs are thought to be mainly involved in suppressing immuneresponses, functioning in part as a “self-check” for the immune systemto prevent excessive reactions. In particular, Tregs are involved inmaintaining tolerance to self-antigens, harmless agents such as pollenor food, and abrogating autoimmune disease.

Tregs are found throughout the body including, without limitation, thegut, skin, lung, and liver. Additionally, Treg cells may also be foundin certain compartments of the body that are not directly exposed to theexternal environment such as the spleen, lymph nodes, and even adiposetissue. Each of these Treg cell populations is known or suspected tohave one or more unique features and additional information may be foundin Lehtimaki and Lahesmaa (Regulatory T cells control immune responsesthrough their non-redundant tissue specific features, FRONTIERS INIMMUNOL., 4(294): 1-10, 2013), the disclosure of which is herebyincorporated in its entirety.

Typically, regulatory T cells are known to require TGF-β and IL-2 forproper activation and development. Blockade of TGF-β signaling has beenshown to result in systemic inflammatory disease as a result of adeficiency of Treg and IL-2 knockout mice have been shown to fail todevelop Treg. TGF-β may be particularly important, as it is known tostimulate Foxp3, the transcription factor that drives differentiation ofT cells toward the Treg lineage.

Tregs are known to produce both IL-10 and TGF-β, both potent immunesuppressive cytokines. Additionally, Tregs are known to inhibit theability of antigen presenting cells (APCs) to stimulate T cells. Oneproposed mechanism for APC inhibition is via CTLA-4, which is expressedby Foxp3⁺Treg. It is thought that CTLA-4 may bind to B7 molecules onAPCs and either block these molecules or remove them by causinginternalization resulting in reduced availability of B7 and an inabilityto provide adequate co-stimulation for immune responses. Additionaldiscussion regarding the origin, differentiation and function of Tregmay be found in Dhamne et al., Peripheral and thymic Foxp3+ regulatory Tcells in search of origin, distinction, and function, 2013, Frontiers inImmunol., 4 (253): 1-11, the disclosure of which is hereby incorporatedin its entirety.

While Tregs are critical for maintaining peripheral tolerance, theirpotent immunoregulatory properties can promote the development ofnumerous types of malignancies (Refs 9, 10) by inhibiting effectorresponses. For many cancers, the presence of large numbers of Treg cellscorrelates with poor outcome (Ref 10 and 98). Furthermore, clinicalevaluation of human breast cancers reveals that the prevalence of Tregamong tumor infiltrating lymphocytes increases with disease stage. Adecrease in the number of breast tumor infiltrating Tregs is positivelyassociated with a pathological response to neoadjuvant chemotherapy.(Refs 59 and 60). Preliminary data reveal that the specific ablation ofTreg in advanced murine breast tumors leads to a significant delay intumor growth and a dramatic reduction in metastatic burden. (Ref 9)

The present invention encompasses the recognition that Tregs integrateexternal local cues to modulate specific segments of theirtranscriptomic program, and they do so by a set of common regulatorsthey share with the ongoing immune response. Without wishing to be boundby any particular theory, the present invention proposes that the tumormicroenvironment also induces a Treg-specific transcriptional program.The present invention further proposes that effective strategies fortargeting tumor-infiltrating Tregs can be developed through anunderstanding of this transcriptional program.

Prior to the present disclosure, data regarding the consequences of thetumor microenvironment on human Treg populations was limited. Asdescribed herein, the present inventors isolated Treg cells, effectorCD4 T cells and CD8 T cells from breast infiltrating duct carcinomas aswell as from normal breast parenchyma and peripheral blood for in depthtranscriptome analysis by RNAseq. Among other things, this work revealedtargets for therapeutic depletion of Treg from the tumormicroenvironment. Specifically, work described herein revealed that CCR8is a useful target specific to tumor-infiltrating Treg cells.

CCR8

CCR8 is a member of the β-chemokine receptor family, which is predictedto be a seven transmembrane protein similar to G protein-coupledreceptors. Chemokines and their receptors are known to be important forthe migration of various cell types into the inflammatory sites.

CCR8 has been reported to play a role in regulation of monocytechemotaxis and thymic cell apoptosis. More specifically, it has beensuggested that CCR8 may contribute to proper positioning of activated Tcells within antigenic challenge sites and specialized areas of lymphoidtissues.

In humans, the gene encoding CCR8 is located in the chemokine receptorgene cluster region 3p22.

Identified ligands of CCR8 include its natural cognate ligand, CCL1 (akaI-309), thymus activation-regulated cytokine (TARC) and macrophageinflammatory protein-1 beta (MIP-1 beta).

CCR8 is preferentially expressed in the thymus, and recent reports haveindicated that its expression is elevated in human cancer tissues,primarily limited to tumor-associated macrophages (see Eruslanov et al,Clin Cancer Res. 19:1670, Epub 2013 Jan. 30). The present disclosureprovides the surprising demonstration that, in fact, CCR8 isspecifically expressed in Treg cells, and more particularly intumor-infiltrating Tregs. The present disclosure specifically teachesthat CCR8 is specifically expressed in tumor-infiltrating Tregs ascompared with other tumor-infiltrating T cell subsets (i.e., tumorinfiltrating CD4 and CD8 T cells), and demonstrates that CCR8 can serveas an effective target to mediate depletion of such tumor-infiltratingTreg cells.

CCR8-Targeting Agents

In light of the provided teaching that CCR8 can effectively be targetedto achieve specific depletion of Treg cells, and particularly oftumor-infiltrating Treg cells, those skilled in the art will appreciatethat any of a variety of appropriate agents may be used to target CCR8and achieve such depletion.

agents that bind specifically to CCR8, or to nucleic acids encoding it.In some embodiments, the present invention provides systems foridentifying and/or characterizing agents that target CCR8 on the surfaceof Treg cells, and most particularly on the surface oftumor-infiltrating Treg cells. In some embodiments, the presentinvention provides systems for identifying and/or characterizing agents(e.g., siRNAs, antisense nucleic acids, CRISPR™ agents, or other nucleicacid binding and/or modifying agents) that bind specifically toCCR8-encoding nucleic acids (e.g., DNA or RNA such as genomic DNA or anytranscript thereof), and block or inhibit its expression (e.g., byblocking or inhibiting transcription, transport, splicing, and/ortranslation of the relevant nucleic acid(s)).

In some embodiments, a CCR8-targeting agent for use in accordance withthe present invention is or comprises a CCR8-specific antibody orantigen-binding fragment thereof. In some embodiments, an CCR8-targetingagent is or comprises an antibody or antigen-binding fragment thereofthat binds to a CCR8 polypeptide found on surfaces of Treg cells, andparticularly on surfaces of Treg cells that have infiltrated a tumor.

In some embodiments, a CCR8-targeting agent that is or comprises anantibody may be or comprise an antibody, or fragment thereof, of anyappropriate isotype, including, for example: IgG (e.g., IgG1, IgG2,IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In some embodiments, anantibody, or fragment thereof, is an IgG isotype, e.g., IgG1 or IgG4.

In some embodiments, a CCR8-targeting agent may be or comprise afull-length antibody. In some embodiments, a CCR8-targeting agent may beor comprise only an antigen-binding fragment (e.g., a Fab, F(ab)2, Fv orsingle chain Fv fragment) of an antibody (e.g., an may lack or besubstantially free of other antibody components). In some embodiments,an a CCR8-targeting agent may be or comprise multiple antigen-bindingcomponents of an antibody (e.g., as in a diabody or zybody). In someembodiments, a CCR8-targeting agent may include one or more CDRs foundin a full-length antibody raised in an organism against the relevantantigen (i.e., against CCR8). In some embodiments, a CCR8-targetingagent may include such CDRs in a different polypeptide context than thatin which they are found in the organism-raised antibody.

In some embodiments, a CCR8-targeting agent may be or comprise anantibody, or fragment thereof, that is monoclonal, recombinant,chimeric, deimmunized, human, humanized, etc as these terms areunderstood in the art.

In some embodiments, a CCR8-targeting agent may be or comprise anantibody Fc region; in some such embodiments, the Fc region may beglycosylated. In some embodiments, such an Fc region may be engineeredso that it is glycosylated and/or otherwise modified in a manner thatmodifies its affinity for, and/or one or more other characteristics ofbinding with Fc receptors on effector cells relative to that observedwith an otherwise comparable or identical Fc region lacking themodification. In some embodiments, a utilized Fc region is engineered tobe modified (e.g., glycosylated) so that its affinity for one or more Fcreceptors on an effector cell is increased relative to that observedwith an otherwise comparable or identical Fc region lacking themodification.

As is known in the art, monoclonal antibodies can be produced by avariety of techniques, including conventional monoclonal antibodymethodology, e.g., the standard somatic cell hybridization technique ofKohler and Milstein, Nature 256: 495, 1975. Polyclonal antibodies can beproduced by immunization of animal or human subjects. See generally,Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1988. Recombinant,chimeric, deimmunized, human, or humanized antibodies can also beproduced using standard techniques, as is known in the art. Techniquesfor engineering and preparing antibodies are described, for example, inU.S. Pat. No. 4,816,567, issued Mar. 28, 1989; U.S. Pat. No. 5,078,998,issued Jan. 7, 1992; U.S. Pat. No. 5,091,513, issued Feb. 25, 1992; U.S.Pat. No. 5,225,539, issued Jul. 6, 1993; U.S. Pat. No. 5,585,089, issuedDec. 17, 1996; U.S. Pat. No. 5,693,761, issued Dec. 2, 1997; U.S. Pat.No. 5,693,762, issued Dec. 2, 1997; U.S. Pat. No. 5,869,619; issued1991; U.S. Pat. No. 6,180,370, issued Jan. 30, 2001; U.S. Pat. No.6,548,640, issued Apr. 15, 2003; U.S. Pat. No. 6,881,557, issued Apr.19, 2005; U.S. Pat. No. 6,982,321, issued Jan. 3, 2006; incorporatedherein by reference. Antibodies described herein can be used, e.g., fordetection (e.g., diagnostic) assays, and/or for therapeuticapplications.

The present disclosure particularly describes use of T-cell depletingagents (e.g., antibody agents) that target CCR8. A variety ofCCR8-specific antibodies are known in the art (see, for example, thosedescribed in Ref 95). Certain CCR8-antagonists, which in certainembodiments may be useful to deplete Treg cells as described herein,have also been reported (See Ref 96). Those skilled in the art willappreciate that known CCR8 antibodies or antagonists, or fragmentsthereof, may be utilized in accordance with the present invention.Alternatively or additionally, those skilled in the art will appreciatethat new CCR8-specific antibodies may be raised, and screened orselected for cell-depleting activity, in accordance withwell-established procedures known in the art (see, for example, Ref 97).

Those skilled in the art will appreciate that useful CCR8-targetingagent for use in accordance with the present invention are not limitedto cell-depleting antibody agents, or even to any antibody agents. Otheragents that specifically interact with CCR8, and particularly with CCR8on surfaces of Treg cells, particularly tumor-infiltrating Treg cells,are useful as described herein. In some embodiments, such agents may beor comprise small molecule agents. In some embodiments, such agents maybe or comprise peptide agents. In some embodiments, such agents may beor comprise one or more CCR8 ligands, or variants thereof.

In some embodiments of the present invention, a CCR8-targeting agent maybe utilized in association with a payload moiety such as a detectablemoiety or a therapeutic (e.g., toxic, anti-proliferative, pro-apoptotic,etc) moiety, in order to detect and/or deplete tumor-infiltrating Tregcells as described herein. In some embodiments, association of aCCR8-targeting entity with a payload is covalent.

For example, in some embodiments, a CCR8-targeting agent as describedherein is provided and/or utilized in association with a payload that isor comprises a detectable moiety such as a radioactive, fluorescent,chemiluminescent, and/or phosphorescent moiety.

In some embodiments, in some embodiments, a CCR8-targeting agent asdescribed herein is utilized in association with a payload that is orcomprises a therapeutic moiety (as is reviewed, for example, withrespect to antibody-drug conjugates in Ducry et al Bioconjugate Chem21:5, 2010; Anderl et ao Methods Mol Biol 1045:51, 2013). To give but afew examples, in some embodiments, a CCR8-targeting agent is providedand/or utilized in association with an anti-proliferative moiety, apro-apoptotic moiety, or with any other moiety whose specific deliveryto tumor-infiltrating Treg cells depletes such cells from the tumor.

In some particular embodiments, a therapeutic moiety may be or comprisea radioactive moiety or another cell-killing moiety (e.g., achemotherapeutic moiety, a cytotoxic moiety, etc). In some embodiments,a cell-killing moiety can be or comprise a small molecule moiety. Insome embodiments, a cell-killing moiety can be or comprise a peptidemoiety. In some embodiments, a cell-killing moiety may be or comprise atoxin or toxin-like (see, for example, those provided by TargaTherapeutics, www.targetherapeutics.com/payloads.html) moiety.

Identifying and/or Characterizing CCR8-Targeting Agents

In some embodiments, the present invention provides systems foridentifying and/or characterizing agents useful in the diagnosis and/ortreatment of cancer, and particularly provides systems for identifyingand/or characterizing agents that bind specifically to CCR8, or tonucleic acids encoding it. In some embodiments, the present inventionprovides systems for identifying and/or characterizing agents thattarget CCR8 on the surface of Treg cells, and most particularly on thesurface of tumor-infiltrating Treg cells. In some embodiments, thepresent invention provides systems for identifying and/or characterizingagents (e.g., siRNAs, antisense nucleic acids, CRISPR™ agents, or othernucleic acid binding and/or modifying agents) that bind specifically toCCR8-encoding nucleic acids (e.g., DNA or RNA such as genomic DNA or anytranscript thereof), and block or inhibit its expression (e.g., byblocking or inhibiting transcription, transport, splicing, and/ortranslation of the relevant nucleic acid(s)). Those skilled in the artare aware of the multiple available technologies for designing,producing, identifying, and/or characterizing effective such nucleicacid binding agents based on the known sequence of CCR8-encoding nucleicacids.

In light of the disclosure provided herein, that CCR8 is specificallyexpressed by Treg cells, and particularly by tumor-infiltrating Tregcells, those skilled in the art will appreciate that agents specificallytargeting CCR8 are useful in a variety of contexts, including asparticularly described herein, and will be aware of a variety of assayformats that can be utilized to identify and/or characterize suchagents.

For example, in some embodiments, CCR8-targeting agents are identifiedand/or characterized as described herein in assays that detect directbinding of such agents to CCR8. In some embodiments, such direct bindingis detected in vitro (e.g., with isolated CCR8 protein); in someembodiments, such direct binding is detected in or on cells (e.g., Tregcells); in some embodiments, such direct binding is detected in or ontissues (e.g., tumor tissues); in some embodiments, such direct bindingis detected in or on an organism (e.g., a model organism, a non-humananimal, or a human).

In some embodiments, useful CCR8-targeting agents as described hereinare identified and characterized based on their ability to detectablybind to cells that express CCR8, and particularly to deplete Treg cells,especially tumor-infiltrating Treg cells.

In some embodiments, useful CCR8-targeting agents as described hereinare identified and characterized based on their ability to deplete(e.g., to inhibit proliferation of and/or to kill) cells that expressCCR8, and particularly to deplete Treg cells, especiallytumor-infiltrating Treg cells.

Those skilled in the art will be aware of a variety of appropriate assayformats for assessing specific binding to CCR8 and/or detection and/ordepletion of CCR8-expressing cells as described herein (see, forexample, MAbs. 2012 July-August; 4(4):445-57; Immunol Rev. 2008 April;222:9-27).

As will be apparent to those skilled in the art, useful assays foridentifying and/or characterizing agents that effectively deplete Tregcells, in some embodiments, can include assays that assess ADCC activityincluding, for example, as described in Clin Cancer Res. 2006 May 1;12(9):2879-87.

In some embodiments, CCR8-targeting agents are identified and/orcharacterized via a process that involves providing a collection of testagents, contacting them with a CCR8-containing system (e.g., an in vitrosystem, a cellular system, a tissue system, and organism) underconditions that permit detection and/or quantification of binding byagents to CCR8 and/or depletion of tumor-infiltrating Treg cells. Insome embodiments, such contacting is performed under conditions that, aswill be understood by those skilled in the art, permit assessment ofCCR8-specific binding and/or Treg depletion. In some embodiments, suchcontacting is performed under conditions that permit comparison with anappropriate reference such as, for example, a positive or negativecontrol agent whose ability to bind specifically to CCR8 and/or todeplete tumor-infiltrating Treg cells is previously known.

Uses

The present disclosure provides data that show CCR8 to be selectivelyexpressed on tumor infiltrating Treg cells as compared to effector Tcells in the tumor as well as normal circulating T cells. These findingsimplicate CCR8 as a therapeutic target to eliminate, inhibit themigration or function of Treg cells, which can, among other things,augment (natural and/or pharmaceutically or otherwise enhanced)anti-tumor immune responses. Alternatively or additionally, thesefindings implicate CCR8 as a diagnostic target for detection and/orcharacterization of tumors infiltrated by Treg cells. In someembodiments, as will be appreciated by those skilled in the art readingthe present disclosure, detection and/or characterization extent and/ornature of Treg infiltration can designate a tumor as likely (or not) tobenefit from certain therapeutic interventions, including as describedherein (e.g., a CCR8-targeting regimen and/or an immunotherapeuticregimen, as described herein). Alternatively or additionally, detectionand/or characterization extent and/or nature of Treg infiltration can beused to monitor effectiveness of a particular therapeutic regimen (e.g.,a CCR8-targeting regimen and/or an immunotherapeutic regimen, asdescribed herein) against a particular tumor, e.g., in a specificsubject. Thus, in various embodiments, the present invention providesboth diagnostic and therapeutic technologies that involve detectingand/or targeting CCR8, and/or Treg cells that express it, in subjectswith tumors.

Extensive preclinical data suggest that Treg cells represent a commonmechanism of immune evasion across most malignancies. Thus, thoseskilled in the art, reading the present disclosure, will appreciate thatCCR8-targeting strategies (e.g., Treg-detection and/or -depletionstrategies as described herein are applicable across a wide range ofmalignancies).

In some embodiments, CCR8-targeting technologies as described herein areuseful in the diagnosis and/or treatment of any of a variety of cancers.The Examples herein specifically demonstrate use of providedtechnologies with respect to breast cancer.

Excluding skin malignancies, breast cancer is the most common canceramong women and the second leading cause of cancer death in women (Ref62). Clinically, pathologic evaluation of breast cancer specimens hasrevealed the prognostic value of certain histopathologic features ofbreast tumors that reflect alterations in the microenvironment includinglymphocytic infiltration, fibrosis, and angiogenesis. While breastcancer has not traditionally been considered an immunogenic tumor, thepresent disclosure appreciates that evidence of tumor infiltratinglymphocytes (TILs) and their subset composition paralleling diseaseprogression suggest that the immune response may be important (Refs: 63and 64). The clinical relevance of tumor infiltrating T cells has beenintensively studied (Ref: 65). An increased ratio of CD4+ to CD8+ Tcells correlates with lymph node metastases and reduced overall survival(Ref: 66). The tumor microenvironment can also influence the recruitmentand regulation of immune cells in breast tumors (Refs: 67 and 68). Highlevels of Treg cells in breast tumors is associated with an invasivephenotype and diminished relapse-free as well as overall survival (Refs:69 and 71). In addition, a substantial decrease in the number of breasttumor infiltrating Treg cells is positively associated with apathological response to neoadjuvant chemotherapy (Ref: 72). Inaccordance with the present invention, breast cancer is a particularcancer of interest for diagnosis and/or treatment with providedtechnologies.

Breast cancer is a heterogeneous disease and is generally classifiedinto three basic therapeutic groups, based on the expression of theestrogen receptor (ER), progesterone receptor (PR), and HER2 (Ref: 73).Triple negative breast cancers (TNBC), also known as basal-like breastcancers, lack expression of ER, PR, and HER2 (Ref: 74). These tumorsaccount for up to 15% of all invasive breast cancers and are frequentlyobserved in patients with BRCA1 germline mutations and of Africanancestry. TNBCs characteristically are densely infiltrated bylymphocytes suggestive of an anti-tumor response, yet are associatedwith a more aggressive clinical course characterized by shorter survivaland higher risk of metastases (Ref: 75). This paradox is hypothesized tobe secondary to effective immune suppression by the tumormicroenvironment with preclinical data implicating Treg cells. Foxp3expression among tumor infiltrating lymphocytes is significantlyassociated with the TNBC subtype of invasive breast cancers (Ref: 76).High levels of Treg cell infiltration of TNBCs is associated shortersurvival (Ref: 77). According to the present invention, TNBC representsa relevant target for diagnosis and/or therapy with providedtechnologies. Moreover, TNBC represents a particularly relevant targetfor use of one or more immunotherapy modalities in combination withCCR8-targeted depletion technologies as described herein, given thatthere is a robust pre-existing infiltrate which can potentially mount ananti-tumor response once the Treg cell-mediated immunosuppression isdiminished through CCR8-mediated Treg cell depletion.

Those skilled in the art, reading the present disclosure, willappreciate that association of Treg cell accumulation in the peripheralblood and tumor infiltrating lymphocytes with clinical outcome is notlimited to patients with breast cancer. As Treg cells represent acentral mechanism of tumor immune evasion, robust tumor infiltration byTreg cells correlates with poor survival in patients affected by manytumor types. Gastric cancer represents one of the most common causes ofcancer-related deaths worldwide (Ref: 78). In metastatic gastric cancer,chemotherapy remains the mainstay of therapy and results in objectiveresponse rates of only 20-40%, with a median overall survival of 8-10months (Ref: 79). This particularly grim prognosis has led to theinvestigation of immunotherapy as a means to improve survival (Ref: 80).Promising results from initial trials suggest that gastric cancer may beamenable to treatment by modulating the immune system. A clinical trialinvestigating the use of autologous cytokine-induced killer cells forlocally advanced gastric cancer patients showed significant improvementsin disease-free and overall survival (Ref: 81). A significant amount ofpreclinical data also suggests that targeting Treg cells in gastriccancer holds therapeutic promise. A study of 133 patients with gastriccancer revealed that high numbers of intratumoral Treg cellssignificantly correlated presence of lymph node metastases and was anindependent factor for adverse overall survival (Ref: 82). Regulatory Tcells from patients with gastric cancer have also been shown to producethe immunosuppressive cytokines IL-4 and IL-10, and inhibit cytokineproduction from CD4+ T cells in vitro (Ref: 83). Treg cell infiltrationof gastric cancers may be in part related to hypoxia, a feature commonto many solid tumors. Treg cell infiltration into gastric cancers ispositively correlated with HIF-1α expression and supernatants fromgastric cancer cells cultured in hypoxic conditions can induce theexpression of Foxp3 in naïve CD4+ T cells via TGF-β1 (Ref: 84). Inaccordance with the present invention, gastric cancer is a target cancerof particular interest for diagnosis and/or therapy with CCR8-targetedtechnologies as provided herein. Moreover, in some embodiments, gastriccancer is a cancer of particular interest for use of providedCCR8-targeted technologies in combination with one or more otherimmunotherapy modalities.

Lung cancer is also a target cancer of particular interest forapplication of provided technologies. Lung cancer is the leading causeof cancer deaths worldwide. The overall 5-year survival rate foradvanced non-small cell lung cancer (NSCLC) is 2%-4%, depending ongeographic location (Ref: 85). Whereas renal cell carcinoma and melanomaare traditionally considered immunogenic, as evidenced by spontaneousregressions and occasional dramatic responses to high-dose IL-2, NSCLChas been notoriously resistant to immunotherapy (Refs 86 and 87).Pre-clinical data suggest that the immune system may have a role in thisdisease. High levels of CD4⁺/CD8⁺ T cells infiltrating resected NSCLCtumors are associated with a favorable prognosis and high levels ofinfiltrating Treg cells are associated with increased risk of relapse(Refs 88 and 89). In early-stage NSCLC the ratio of Treg cells to CD3+TILs correlates with disease specific survival and can reliablydistinguish patients with tumors who are at high risk for recurrence(Refs 90 and 91). Genetic evidence pointing to the role of Treg cells inthe risk of developing NSCLC was documented by determining the presenceof a single nucleotide polymorphism (SNP) associated with Graves diseasein a cohort of patients with NSCLC and healthy controls. The studydemonstrated a significant association of this SNP with a risk ofdeveloping NSCLC (Ref 92). Recently there have been significantbreakthroughs in harnessing the immune system through the use ofcheckpoint blockade inhibitory antibodies to treat NSCLC. In a Phase Idose escalation study of 207 patients treated with an anti-PD-L1monoclonal antibody, a response rate of 10% was observed in patientswith NSCLC (Ref 93). Another Phase I dose-escalation study of 296patients treated with an anti-PD-1 antibody reported a response rate of18% in patients with NSCLC (Ref 94). The present disclosure provides theinsight that the association of Treg cells with the clinical course ofNSCLC patients as well as the significant pool of proof-of-principleclinical data that immune modulation can yield therapeutic responses inthis disease, depleting Treg cells is a promising therapeutic modalityfor lung cancer patients, and particularly for NSCLC patients. Moreover,in some embodiments, lung cancer, including specifically NSCLC, is acancer of particular interest for use of provided CCR8-targetedtechnologies in combination with one or more other immunotherapymodalities.

Combination

The present invention appreciates that, in many embodiments, provideddiagnostic and/or therapeutic compositions and/or strategies that targetCCR8 (e.g., by utilizing CCR8-targeting agents) may be combined with oneor more other therapeutic and/or diagnostic modalities useful in thetreatment of cancer. Those skilled in the art will be well aware ofapproved or otherwise appropriate such modalities, taking intoconsideration the type of cancer involved in any particular instance,and/or the nature, characteristics, and/or history of the relevantsubject.

In some embodiments, provided CCR8-targeting strategies are combinedwith forms of treatment including but not limited to pharmacotherapy,chemotherapy, mesotherapy, medical devices, surgery, gene therapy,hormone therapy, radiotherapy, phototherapy, electrotherapy,thermotherapy, and cryotherapy. In some embodiments, provided CCR8targeting strategies are combined with biologics, cells, proteins,steroids, hormones, cytokines, enzymes, peptides, polypeptides, aminoacids, nucleic acids, DNA, RNA, mRNA, tRNA, siRNA, dsRNA, DNA vaccines,antibodies, monoclonal antibodies, polyclonal antibodies, antibody-drugconjugates, antivirals, antibiotics, antifungals and any conjugatesthereof.

In some embodiments, provided CCR8 targeting strategies are combinedwith anti-inflammatory agents to treat cancer. Anti-inflammatory agentsinclude both steroids and non-steroidal anti-inflammatory drugs (NSAID).In some embodiments, provided CCR8 targeting strategies are combinedwith steroids, including but not limited to glucocorticoids andcorticosteroids. In some embodiments, provided CCR8 targeting strategiesare combined with non-steroidal anti-inflammatory drugs, including butnot limited to ibuprofen, aspirin, naproxen sodium, celecoxib, sulindac,oxaprozin, salsalate, diflunisal, piroxicam, indomethacin, etodolac,meloxicam, naproxen, nabumetone, tromethamine, diclofenac, esomeprazole,and acetaminophen.

In some embodiments, provided CCR8 targeting strategies are combinedwith medical imaging modalities to treat and/or monitor cancer. In someembodiments, provided CCR8 targeting strategies are combined withmedical imaging modalities including but not limited toechocardiography, thermography, tomography, photoacoustic imaging,ultrasound, magnetic resonance imaging, nuclear medicine, elastography,positron emission tomography, computed tomography, and fluorescencetomography.

In some embodiments, CCR8 targeting strategies as described herein arecombined with other strategies that also specifically target Treg cells,and particularly with strategies that specifically targettumor-infiltrating Treg cells. For example, in some particularembodiments, diagnostic and/or therapeutic compositions and/orstrategies utilizing CCR8-targeting agents may be combined with one ormore other therapeutic and/or diagnostic modalities that also targetTregs for depletion (as reviewed, for example, in Pere et al,Oncoimmunology 1(3):326, 2012 May 1).

To give but a few examples, certain chemotherapeutic regimens arethought to reduce Treg by promoting dendritic cell maturation (see, forexample, Zitvogel et al Nat Rev Clin Oncol. 8:151, 2011). Alternativelyor additionally, modalities that destroy proliferating cells, may shiftthe balance of T cells in tumor environments toward T effector cells ascompared with Tregs. In some embodiments, metronomic dosing of suchanti-proliferative agents may achieve selective reduction of Tregpopulations (see for example Polak & Turk Nature. 249:654, 1974;Ercolini et al J Exp Med. 201:1591, 2005; Chen et al. Mol Ther. 18:1233,2010).

Alternatively or additionally, in some embodiments, the presentinvention contemplates combination of provided diagnostic and/ortherapeutic compositions and/or strategies utilizing CCR8-targeting withone or more other modalities that also targets a Treg-associated markeror markers. Representative such markers include for example, CD25,CTLA-5. GITR. OX40, one or more TLR ligands (e.g., TLR4, TLRS, TLR7,and/or TLR8), CD39, CD73, Foxp3, CCL17, CCR4, CCL22, CCR7, and/or CCR5.

CTLA-4 is expressed on both regulatory and activated T cells, andanti-CTLA-4 strategies have been proposed as potential approaches fordepleting Treg cells or otherwise improving and/or promoting anti-tumorimmune reactions (see, for example, Peggs et al J Exp Med 206:1717,2009).

GITR is constitutively expressed by Treg and is also detected, albeit atlower levels, on CD4⁺ and CD8⁺ effector T cells. Agonistic antibodies toeither GITR or GITR ligand have been reported to 1) suppress Tregactivity; 2) enhance proliferation of effector T cells; and 3) improveeffector cells' ability to resist Treg-mediated suppression (see, forexample, Turk et al J Exp Med 200:771, 2004; Ko et al J Exp Med.202:885, 2005; Stephens et al J Immunol. 173:5008, 2004).

OX40 is a costimulatory molecule of the TNF receptor family that isconstitutively expressed on Treg and transiently expressed on activatedeffector T cells. Use of agnonstic anti-OX40 antibodies has beenreported to inhibit Treg suppressive activity, and to reduce tumorgrowth (Valzasina et al Blood. 105:2845, 2005; Piconese et al. J ExpMed. 205:825, 2008).)

Various TLRs (including specifically TLR4, TLR5, TLR7 and TLR8) areexpressed bt Treg cells, and activation of at least TLR 8 has been shownto inhibit Treg function and enhance in vivo tumor immunity (Peng et alScience. 309:1380, 2005).

Treg numbers and/or function can also be suppressed by inhibitingcatabolism of adenine nucleotides (ATP, ADP and AMP) by extracellularectonucleotidases, CD39 and CD73, for example through use of adenosineinhibitors and/or adenosine receptor antagonists (Blackburn et al HandbExp Pharmacol. 215, 2009). Inhibition of CD39 with enzymatic inhibitorshas been reported to Treg function and improve certain effects ofchemotherapy (Michaud et al Science. 334:1573, 2011).

Inhibition of Foxp3 has been reported to impair Treg activity (Casareset al. J Immunol. 185:5150, 2010), and may in some embodiments be usefulin accordance with the present invention.

Another strategy to inhibit and/or deplete Tregs, for example incombination with inventive strategies as described herein, is to targetchemokine/chemokine receptor molecules (e.g., CCL17/CCL22-CCR4 axis)that are involved in Treg trafficking and/or in T cell suppression ofeffector cell activity. Tumor cells and their microenvironment attractTreg by the secretion of CCL22 (22), and a correlation has been reportedbetween the presence of tumor-infiltrating Treg and CCL22 in breastcancer (Gobert et al Cancer Res. 69:2000, 2009). In a murine model, ithas been shown that monoclonal antibodies specific for CCL22significantly reduce the migration of Treg into ovarian tumors (Curielet al Nat Med. 10:942, 2004). Recently, small molecule antagonists ofCCR4 have been shown to prevent the interaction of CCL22/CCL17 withtheir receptor and to inhibit the recruitment of Treg mediated by CCL22and CCL17. The present disclosure specifically demonstrates expressionof CCR4 in tumor-infiltrating Tregs; in some embodiments, CCR8 and CCR4are targeted in combination (e.g., simultaneously).

Other chemokine receptors such as CCR7 and CCR5 may also play a role inTreg migration; administration of a CCR5 inhibitor has been reported toreduce Treg migration into tumors, and to increase effectiveness ofimmunotherapy in reducing tumors (see, for example, Tan et al. JImmunol. 182:1746, 2009; Conforti et al. Cancer Res. 70:490, 2010).

In some particular embodiments, diagnostic and/or therapeuticcompositions and/or strategies utilizing CCR8-targeting agents may becombined with one or more other therapeutic and/or diagnostic modalitiesthat inhibit angiogenesis.

In some particular embodiments, diagnostic and/or therapeuticcompositions and/or strategies utilizing CCR8-targeting agents may becombined with one or more other therapeutic and/or diagnostic modalitiesthat target one or more tyrosine kinases. For example, it has beendemonstrates that in both mouse and human gastrointestinal carcinomas,use of tyrosine kinase inhibitors (e.g., imatinib mesylate, dasatinib,temozolomide, etc) can reduce Treg cell numbers in tumors, resulting inincreased Natural Killer (NK) cell functions effective against them(see, for example, Balachandran et al Nat Med. 17:1094, 2011; Banissi etal Cancer Immunol Immunother. 58:1627, 2009; Delahaye et al. Nat Med.17:700, 2011).

In some particular embodiments that involve combination targeting of twoor more markers such as, for example, two or more Treg-associatedmarkers, the present invention contemplates use of multifunctionaltargeting agents as are known in the art such as, for example,multi-functional antibody formats including but not limited tomultifunctional (e.g., bifunctional antibodies, zybodies,glycoengineered antibodies, etc).

The present disclosure appreciates that accumulating evidencedemonstrates that Treg cells utilize components of the specific immuneresponse in which they are acting to suppress the immune responseitself. For example, expression of the transcription factor Irf4 isrequired for Tregs to be able to suppress TH2 responses, and Irf4controls a module of the Foxp3 program that contains 20% of its targets(Ref. 15). Similarly CXCR3, a target of Tbet, is required for control ofTH1 responses (Ref. 16), and Stat3 for TH17 responses (Ref. 17). Withoutwishing to be bound by any particular theory, the present disclosureobserves that observations suggest that Tregs integrate external localcues to modulate specific segments of their transcriptomic program, andfurthermore that they do so by a set of common regulators they sharewith the ongoing immune response.

The present disclosure further provides the insight that, because Tregcells may be critical mediators of the immunosuppressivemicroenvironment, depleting or ablating Treg as described herein mayconvert tumor subtypes classically considered “non-immunogenic” to astate where immune intervention is possible. The present inventiontherefore specifically teaches, in some embodiments, the treatment ofcancer by depletion of Treg cells (e.g., through use of CCR8-targetingagents as described herein) in combination with one or moreimmunomodulatory modalities of cancer therapy such as, for example,antibody blockage of inhibitory molecules, adoptive T cell transfer,and/or vaccination or other strategies. In some embodiments of thepresent invention, CCR8-targeting is combined with one or more suchstrategies to detect, characterize, and/or treat cancer.

The present disclosure specifically contemplates combination of providedCCR8-targeting technologies with ionizing radiation therapy. Ionizingradiation therapy is widely used for the management of certain cancers,particularly including breast cancer, and its advantages derive from itsability to induce cell death and proliferation arrest, depending on thecontext. More recently, it has been suggested that radiotherapy has theadded benefit of immune modulation (see, for example, Formenti et alLancet Onc. 10:718, July 2009) contributing to enhanced antigen anddanger signal release from dying cancer cells and antigen presentationfrom dendritic cells, as well as down-regulating antigens upon reductionof tumor mass, which reduces the chances of tolerance induction.Specific rational for a combinatorial approach with Treg ablation asdescribed herein is the known relative radio-resistance of Treg cells,discovered when exploring the reasons behind the inability to transmitdisease through scurfy bone marrow transfer into lethally irradiatedhosts (see, for example, Komatsu et al Proc. Natl. Acad. Sci USA104:8959, May 22, 2007. Current clinical outcomes might be significantlyimproved by combination of Treg depletion strategies with radiation, andpossibly chemotherapy or targeted therapies against molecular drivers ofoncogenesis.

Pharmaceutical Compositions

In some embodiments, the present invention provides pharmaceuticalcompositions comprising one or more provided agents (e.g., CCR-8targeting agents) together with one or more pharmaceutically acceptableexcipients.

In some embodiments, provided pharmaceutical compositions may beprepared by any appropriate method, for example as known or hereafterdeveloped in the art of pharmacology. In general, such preparatorymethods include the step of bringing a provided CCR8 targeting agentinto association with one or more pharmaceutically acceptableexcipients, and then, if necessary and/or desirable, shaping and/orpackaging the product into an appropriate form for administration, forexample as or in a single- or multi-dose unit.

In some embodiments, compositions may be prepared, packaged, and/or soldin bulk, as a single unit dose, and/or as a plurality of single unitdoses. As used herein, a “unit dose” is a discrete amount of thepharmaceutical composition comprising a predetermined amount of one ormore provided CCR8 targeting agent. The amount of the provided CCR8targeting agent is generally equal to the dosage of the provided CCR8targeting agent which would be administered to a subject and/or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

In some embodiments, provided pharmaceutical compositions arespecifically formulated for mucosal delivery (e.g., oral, nasal, rectalor sublingual delivery). In some embodiments, pharmaceuticalcompositions are specifically formulated for oral delivery as beingconjugated to starch and mixed with food.

In some embodiments, appropriate excipients for use in providedpharmaceutical compositions may, for example, include one or morepharmaceutically acceptable solvents, dispersion media, granulatingmedia, diluents, or other liquid vehicles, dispersion or suspensionaids, surface active agents and/or emulsifiers, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants, disintegrating agents, binding agents, preservatives,buffering agents and the like, as suited to the particular dosage formdesired. Alternatively or additionally, pharmaceutically acceptableexcipients such as cocoa butter and/or suppository waxes, coloringagents, coating agents, sweetening, flavoring, and/or perfuming agentscan be utilized. Remington's The Science and Practice of Pharmacy,21^(st) Edition, A. R. Gennaro (Lippincott, Williams & Wilkins,Baltimore, Md., 2005; incorporated herein by reference) disclosesvarious excipients used in formulating pharmaceutical compositions andknown techniques for the preparation thereof.

In some embodiments, an appropriate excipient is at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% pure. In someembodiments, an excipient is approved by United States Food and DrugAdministration. In some embodiments, an excipient is pharmaceuticalgrade. In some embodiments, an excipient meets the standards of theUnited States Pharmacopoeia (USP), the European Pharmacopoeia (EP), theBritish Pharmacopoeia, and/or other International Pharmacopoeia.

In some embodiments, liquid dosage forms (e.g., for oral and/orparenteral administration) include, but are not limited to, emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to provided CCR8 targeting agent(s), liquid dosage forms maycomprise inert diluents commonly used in the art such as, for example,water or other solvents, solubilizing agents and emulsifiers such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and/or perfuming agents. In certain embodimentsfor parenteral administration, compositions are mixed with solubilizingagents such a CREMOPHOR®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

In some embodiments, injectable preparations, for example, sterileaqueous or oleaginous suspensions, may be formulated according to knownmethods using suitable dispersing agents, wetting agents, and/orsuspending agents. Sterile liquid preparations may be, for example,solutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed, for example, are water, Ringer's solution, U.S.P., andisotonic sodium chloride solution. Sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono- ordiglycerides. Fatty acids such as oleic acid can be used in thepreparation of liquid formulations.

Liquid formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In some embodiments, one or more strategies may be utilized prolongand/or delay the effect of a provided CCR8 targeting agent afterdelivery.

In some embodiments, provided pharmaceutical compositions may beformulated as suppositories, for example for rectal or vaginal delivery.In some embodiments, suppository formulations can be prepared by mixingutilizing suitable non-irritating excipients such as cocoa butter,polyethylene glycol or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in thebody (e.g., in the rectum or vaginal cavity) and release the providedCCR8 targeting agent.

In some embodiments, solid dosage forms (e.g., for oral administration)include capsules, tablets, pills, powders, and/or granules. In suchsolid dosage forms, the provided CCR8 targeting agent(s) may be mixedwith at least one inert, pharmaceutically acceptable excipient such assodium citrate or dicalcium phosphate and/or fillers or extenders (e.g.,starches, lactose, sucrose, glucose, mannitol, and silicic acid),binders (e.g., carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.,glycerol), disintegrating agents (e.g., agar, calcium carbonate, potatostarch, tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g., paraffin), absorptionaccelerators (e.g., quaternary ammonium compounds), wetting agents(e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g.,kaolin and bentonite clay), and lubricants (e.g., talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate), and mixtures thereof. In the case of capsules, tablets andpills, the dosage form may comprise buffering agents.

In some embodiments, solid compositions of a similar type may beemployed as fillers in soft and/or hard-filled gelatin capsules usingsuch excipients as lactose or milk sugar as well as high molecularweight polyethylene glycols and the like. The solid dosage forms oftablets, dragees, capsules, pills, and granules can be prepared withcoatings and shells such as enteric coatings and other coatings wellknown in the pharmaceutical formulating art.

Exemplary enteric coatings include, but are not limited to, one or moreof the following: cellulose acetate phthalate; methylacrylate-methacrylic acid copolymers; cellulose acetate succinate;hydroxy propyl methyl cellulose phthalate; hydroxy propyl methylcellulose acetate succinate (hypromellose acetate succinate); HP55;polyvinyl acetate phthalate (PVAP); methyl methacrylate-methacrylic acidcopolymers; methacrylic acid copolymers, cellulose acetate (and itssuccinate and phthalate version); styrol maleic acid co-polymers;polymethacrylic acid/acrylic acid copolymer; hydroxyethyl ethylcellulose phthalate; hydroxypropyl methyl cellulose acetate succinate;cellulose acetate tetrahydrophtalate; acrylic resin; shellac, andcombinations thereof.

In some embodiments, solid dosage forms may optionally compriseopacifying agents and can be of a composition that they release theprovided CCR8 targeting agent(s) only, or preferentially, in a certainpart of the intestinal tract, optionally, in a delayed manner. Examplesof embedding compositions which can be used include polymeric substancesand waxes. Solid compositions of a similar type may be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugar as well as high molecular weight polyethyleneglycols and the like.

In some embodiments, the present invention provides compositions fortopical and/or transdermal delivery, e.g., as a cream, liniment,ointment, oil, foam, spray, lotion, liquid, powder, thickening lotion,or gel. Particular exemplary such formulations may be prepared, forexample, as products such as skin softeners, nutritional lotion typeemulsions, cleansing lotions, cleansing creams, skin milks, emollientlotions, massage creams, emollient creams, make-up bases, lipsticks,facial packs or facial gels, cleaner formulations such as shampoos,rinses, body cleansers, hair-tonics, or soaps, or dermatologicalcompositions such as lotions, ointments, gels, creams, liniments,patches, deodorants, or sprays.

In some embodiments, provided compositions are stable for extendedperiods of time, such as 1 week, 2 weeks, 1 month, 2 months, 6 months, 1year, 2 years, 3 years, or more. In some embodiments, providedcompositions are easily transportable and may even be sent viatraditional courier or other package delivery service. Accordingly, someembodiments may be useful in situations of disease outbreak, such asepidemics, or attacks with biological agents at least in part due totheir ability to be stored for long periods of time and transportedquickly, easily, and safely. Such attributes may allow for rapiddistribution of provided compositions to those in need.

In some embodiments, it may be advantageous to release CCR8 targetingagent(s), at various locations along a subject's gastrointestinal (GI)tract. In some embodiments, it may be advantageous to release CCR8targeting agent(s), for example, an agent, in a subject's mouth as wellas one or more locations along the subject's GI tract. In someembodiments, it may be advantageous to release CCR8 targeting agent(s),for example, an agent, in a subject's GI tract, including but notlimited to the stomach, intestines, and colon. Accordingly, in someembodiments, a plurality of provided compositions (e.g., two or more)may be administered to a single subject to facilitate release of CCR8targeting agent(s) at multiple locations. In some embodiments, each ofthe plurality of compositions has a different release profile, such asprovided by various enteric coatings, for example. In some embodiments,each of the plurality of compositions has a similar release profile. Insome embodiments, the plurality of compositions comprises one or moreCCR8 targeting agents. In some embodiments, each of the plurality ofadministered compositions comprises a different CCR8 targeting agent. Insome embodiments, each of the plurality of compositions comprises thesame CCR8 targeting agent.

Dosing

It is contemplated that any of a variety of dosing regimens may be usedin accordance with various embodiments. In some embodiments, the step ofstimulating comprises administering at least two doses of a CCR8targeting agent, separated by a period of time. In some embodiments, thestep of stimulating comprises administering at least three, four, five,six or more than six doses of a CCR8 targeting agent, each separated bya period of time. In some embodiments, the period of time between eachadministration is the same. In some embodiments, the period of timebetween each administration is different. In some embodiments, theperiod of time between doses may be 1 minute, 15 minutes, 30 minutes, 1hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours,96 hours, 1 week, 2 weeks, or 1 month. In some embodiments, the periodof time between doses is greater than 1 month. In some embodiments, eachdose is administered substantially simultaneously (e.g., sequentially).

According to various embodiments comprising administration of two ormore doses of a CCR8 targeting agent, the dose of CCR8 targeting agentmay vary according to sound medical judgment. In some embodiments, eachdose of a CCR8 targeting agent is the same. In some embodiments, eachdose of a CCR8 targeting agent may vary from one or more other doses.

In some embodiments, a CCR8 targeting agent is administered at a doseequal to or approximating a therapeutically effective amount. In someembodiments, a therapeutically effective amount of a CCR8 targetingagent may be an amount ranging from about 0.001 to about 1,000 mg/kg. Insome embodiments, a therapeutically effective amount may be, forexample, about 0.001 to 500 mg/kg weight, e.g., from about 0.001 to 400mg/kg weight, from about 0.001 to 300 mg/kg weight, from about 0.001 to200 mg/kg weight, from about 0.001 to 100 mg/kg weight, from about 0.001to 90 mg/kg weight, from about 0.001 to 80 mg/kg weight, from about0.001 to 70 mg/kg weight, from about 0.001 to 60 mg/kg weight, fromabout 0.001 to 50 mg/kg weight, from about 0.001 to 40 mg/kg weight,from about 0.001 to 30 mg/kg weight, from about 0.001 to 25 mg/kgweight, from about 0.001 to 20 mg/kg weight, from about 0.001 to 15mg/kg weight, from about 0.001 to 10 mg/kg weight. In some embodiments,the therapeutically effective amount described herein is provided in onedose. In some embodiments, the therapeutically effective amountdescribed herein is provided in one day.

In some embodiments, a therapeutically effective dosage amount may be,for example, about 0.0001 to about 0.1 mg/kg weight, e.g. from about0.0001 to 0.09 mg/kg weight, from about 0.0001 to 0.08 mg/kg weight,from about 0.0001 to 0.07 mg/kg weight, from about 0.0001 to 0.06 mg/kgweight, from about 0.0001 to 0.05 mg/kg weight, from about 0.0001 toabout 0.04 mg/kg weight, from about 0.0001 to 0.03 mg/kg weight, fromabout 0.0001 to 0.02 mg/kg weight, from about 0.0001 to 0.019 mg/kgweight, from about 0.0001 to 0.018 mg/kg weight, from about 0.0001 to0.017 mg/kg weight, from about 0.0001 to 0.016 mg/kg weight, from about0.0001 to 0.015 mg/kg weight, from about 0.0001 to 0.014 mg/kg weight,from about 0.0001 to 0.013 mg/kg weight, from about 0.0001 to 0.012mg/kg weight, from about 0.0001 to 0.011 mg/kg weight, from about 0.0001to 0.01 mg/kg weight, from about 0.0001 to 0.009 mg/kg weight, fromabout 0.0001 to 0.008 mg/kg weight, from about 0.0001 to 0.007 mg/kgweight, from about 0.0001 to 0.006 mg/kg weight, from about 0.0001 to0.005 mg/kg weight, from about 0.0001 to 0.004 mg/kg weight, from about0.0001 to 0.003 mg/kg weight, from about 0.0001 to 0.002 mg/kg weight.The effective dose for a particular individual can be varied (e.g.,increased or decreased) over time, depending on the needs of theindividual.

Routes of Administration

In some embodiments, provided CCR8 targeting agents and compositionscomprising the same may be formulated for any appropriate route ofdelivery. In some embodiments, provided CCR8 targeting agents andcompositions comprising the same may be formulated for any route ofdelivery, including, but not limited to, bronchial instillation, and/orinhalation; buccal, enteral, interdermal, intra-arterial (IA),intradermal, intragastric (IG), intramedullary, intramuscular (IM),intranasal, intraperitoneal (IP), intrathecal, intratrachealinstillation (by), intravenous (IV), intraventricular, mucosal, nasalspray, and/or aerosol, oral (PO), as an oral spray, rectal (PR),subcutaneous (SQ), sublingual; topical and/or transdermal (e.g., bylotions, creams, liniments, ointments, powders, gels, drops, etc.),transdermal, vaginal, vitreal, and/or through a portal vein catheter;and/or combinations thereof. In some embodiments, the present inventionprovides methods of administration of CCR8 targeting agents andcompositions comprising the same via mucosal administration. In someembodiments, the present invention provides methods of administration ofCCR8 targeting agents and compositions comprising the same via oraladministration.

In some embodiments, provided CCR8 targeting agents and compositionscomprising the same may be formulated for oral delivery. In someembodiments, provided CCR8 targeting agents and compositions comprisingthe same may be formulated for topical delivery.

Kits

In some embodiments, the present invention further provides kits orother articles of manufacture which contain one or more CCR8 targetingagents or formulations containing the same, and provides instructionsfor its reconstitution (if lyophilized) and/or use. In some embodiments,a kit may comprise (i) at least one provided CCR8 targeting agent orcomposition comprising the same; and (ii) at least one pharmaceuticallyacceptable excipient; and, optionally, (iii) instructions for use.

Kits or other articles of manufacture may include a container, asyringe, vial and any other articles, devices or equipment useful inadministration (e.g., subcutaneous, by inhalation). Suitable containersinclude, for example, bottles, vials, syringes (e.g., pre-filledsyringes), ampules, cartridges, reservoirs, or lyo-jects. The containermay be formed from a variety of materials such as glass or plastic. Insome embodiments, a container is a pre-filled syringe. Suitablepre-filled syringes include, but are not limited to, borosilicate glasssyringes with baked silicone coating, borosilicate glass syringes withsprayed silicone, or plastic resin syringes without silicone.

Typically, the container may holds formulations and a label on, orassociated with, the container that may indicate directions forreconstitution and/or use. For example, the label may indicate that theformulation is reconstituted to concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor, for example, subcutaneous administration.

In some embodiments, a container may contain a single dose of a stableformulation containing one or more CCR8 targeting agents. In variousembodiments, a single dose of the stable formulation is present in avolume of less than about 15 ml, 10 ml, 5.0 ml, 4.0 ml, 3.5 ml, 3.0 ml,2.5 ml, 2.0 ml, 1.5 ml, 1.0 ml, or 0.5 ml. Alternatively, a containerholding the formulation may be a multi-use vial, which allows for repeatadministrations (e.g., from 2-6 administrations) of the formulation.Kits or other articles of manufacture may further include a secondcontainer comprising a suitable diluent (e.g., BWFI, saline, bufferedsaline).

Upon mixing of the diluent and the formulation, the final proteinconcentration in the reconstituted formulation will generally be atleast 1 mg/ml (e.g., at least 5 mg/ml, at least 10 mg/ml, at least 20mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least75 mg/ml, at least 100 mg/ml).

Kits or other articles of manufacture may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use. In some embodiments, kits or other articlesof manufacture may include an instruction for self-administration.

In some embodiments, kits include multiple (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) doses ofprovided CCR8 targeting agents and/or compositions comprising the same.In some embodiments, kits include multiple (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) populationsof provided CCR8 targeting agents and/or compositions comprising thesame having different functional elements (e.g., CCR8 targeting agents).In some embodiments, multiple populations of provided CCR8 targetingagents and/or compositions comprising the same are packaged separatelyfrom one another in provided kits. In some embodiments, provided kitsmay include provided compositions and one or more other therapeuticagents intended for administration with the provided compositions.

EXAMPLES Example 1 Targeting CCR8 to Specifically DepleteTumor-Infiltrating Tregs

The present Example describes identification of CCR8 as a specificmarker of tumor infiltrating Tregs and as a target against whichtherapeutic strategies (e.g., depleting antibodies, can and should bedirected). Specifically, the present Example demonstrates that CCR8 isspecifically expressed in tumor-infiltrating Tregs as compared withother tumor infiltrating T cell subsets (i.e., CD4 and CD8 T cells.

Among other things, the present work identifies the source of a problemin certain prior efforts to identify and/or characterize differentiallyexpressed targetable genes in tumor-associated immune cell subsets.Specifically, the present work appreciates that, in the tumormicroenvironment, not only does immune cell composition vary from tumorto tumor (Ref: 42), but each cell type within a tumor depends oninteractions with other cells for its recruitment, survival, orfunction. Large-scale studies that collect genomic or transcriptomicdata from total tissue produce variable data that is difficult tointerpret without considering the microenvironmental context. In silicodissection of mixed cell samples using raw data has been utilized todeconvolve expression data, but underdetection of gene expressionsignatures and their relative functional contribution is still a majorproblem (Ref: 43).

We isolated Treg cells, effector CD4 T cells and CD8 T cells from breastinfiltrating duct carcinomas as well as normal breast parenchyma andperipheral blood for in depth transcriptome analysis by RNAseq.

Tumor as well as corresponding normal tissue taken far from the primarytumor, or from contralateral prophylactic mastectomies, was harvested bythe tumor Procurement Service (TPS) at Memorial Sloan Kettering CancerCenter. Tumor and normal tissue were cut in fine pieces and transferredto a 40 μm nylon mesh cell strainer immersed in 5 ml PBS buffer in aPetri dish. The tissue was be gently mashed with the end of a plunger tocreate a single cell suspension. A second filtration through a 40 μmnylon mesh was carried out. This procedure was repeated as necessary ordesirable to wash the filters utilized and obtain maximal recovery.Cells were pelleted by spinning at 1000 rpm for 10 minutes at 4° C.Supernatant was aspirated carefully and a second wash was performed. Thecell pellet resulting from this procedure was resuspended and stainedwith fluorescent antibodies for analysis by flow cytometry. Human Tregswere defined as CD3⁺CD4⁺CD45RA⁻CD45RO⁺CD25⁺ (>95% Foxp3⁺, FIG. 4);effector CD4⁺ cells were defined as CD3⁺CD4⁺CD45RA⁻CD45RO⁺CD25⁻>95%Foxp3⁻, FIG. 4), and effector CD8⁺ T cells were defined as CD3⁺CD8⁺.Fluorescence-activated cell sorting (FACS) was used to obtain purepopulations of these TIL subsets. Cells were sorted into Trizol LS™, andwere stored at −80° C. until RNA extraction.

From the isolated cells, total RNA was extracted using Trizol LS™reagent. cDNA was generated from 3 ng of rRNA depleted RNA using theSMARTer Universal Low Input Kit (Clontech) according to manufacturesguidelines. First strand synthesis was initiated by a 5′-modified randomhexamer utilizing reverse transcriptase with terminal transferaseactivity resulting in the addition of a few additional nucleotides atthe 3′ end of the newly synthesized cDNA. The newly-formed cDNA overhangbase paired with the SMARTer oligonucleotide, thus creating an extendedtemplate for the RT and yielding modified cDNA fragments. Subsequentsecond strand synthesis and cDNA amplification were driven from theuniversal priming sites thus generating an unbiased cDNA population.

After ribogreen quantification and quality control of AgilentBioAnalyzer (RIN>7), resulting cDNA was sheared using the Covaris toachieve fragments in the range of 200 bp. Fragmented sample quality andyield were evaluated using Agilent BioAnalyzer. Fragmented material wasused for library preparation according to the Ion ChIP-Seq Kit startingwith the end-repair process (Life Technologies), with 12 to 16 cycles ofPCR. The resulting barcoded samples were loaded onto template-positiveIon PI™ Ion Sphere™ Particles (ISPs) using the Ion One Touch system IIand Ion PI™Template OT2 200kit v2 Kit (Life Technologies). Enrichedparticles were sequenced on a Proton sequencing system using the 200 bpversion 2 chemistry. On average of 70 to 80 million reads were generatedper samples. The sequence data were processed and analyzed with theassistance of the Bioinformatics core facility at the Sloan KetteringInstitute.

Our initial RNAseq data reveled CCR8 to be highly expressed in tumorinfiltrating Treg and minimally expressed on other immune cellsinfiltrating the tumor (FIG. 5). In addition there was is very littleexpression of CCR8 on circulating blood Treg cells.

We confirmed the mRNA expression patterns of CCR8 by measuring CCR8expression on the protein level by flow cytometry (FIG. 6). We foundCCR8 to be preferentially expressed on Treg cells infiltrating humanbreast and colon cancer. In addition there was less of a differentialexpression pattern observed in normal tissue infiltrating T cells.Similarly there was little expression of CCR8 on circulating normalblood T cells. We therefore defined CCR8 as a useful target fordepleting tumor-infiltrating Treg cells.

Furthermore, we found that there was significant overlap in expressionof CCR4 and CCR8 expression on tumor infiltrating Treg cells, yet therewere also Treg cells that expressed either chemokine receptor alone(FIG. 7). In some embodiments, the present disclosure contemplates acombination strategy in which both CCR8 and CCR4 are targeted to depletetumor infiltrating Treg cells Such a strategy, in accordance with thepresent invention, would be able to effectively target 99% of tumorinfiltrating Treg cells.

Example 2 Initial Studies Targeting Other Treg Surface Markers

The present Example describes initial studies targeting other markerspresent on the surface of Treg cells.

CTLA-4, PD-1 and its ligand PD-L1, like CCR8, are present in largeamounts on the surface of Treg cells (21). Recently, CTLA-4 andPD-1/PD-L1 antibody-mediated blockade have been proven a viableimmunotherapeutic strategy to treat solid tumors in pre-clinical andclinical settings.

The present Example describes studies to test whether combination withCTLA-4 or PD-1 checkpoint blockade might enhance the therapeutic effectof Treg cell ablation in our oncogene-driven breast cancer model.Blockade of either one of these pathways by CTLA-4 or PD-1 or PD-L1 or acombination of PD-1 and PD-L1 antibodies had no significant effect onthe growth of PyMT-driven orthotopic tumors (data not shown). Theseresults suggest that efficient targeting of Treg cells is necessary andsufficient to achieve an effective immunotherapeutic response to thegrowing tumor in this model of oncogene-dependent cancer.

Example 3 Confirmation of CCR8 Expression on Tumor Infiltrating Tregs

The present Example further documents the role of CCR8 expression ontumor infiltrating Tregs. For example, the present Example documentseffects of CCR8 Treg expression on tumor volume, metastases, andproliferation.

The consequence of CCR8 expression on tumor infiltrating Treg cells wasexamined using a mixed bone marrow chimera experimental strategy (FIG.8). With this approach we were able to develop two groups of mice, onewith normal Treg cells and another with CCR8 deficient Treg cells.Carcinoma cells from C57BL/6 mice expressing a transgene encoding thePyMT oncogene under control of the MMTV promoter were implanted in themammary fat pads of these two groups of mice. Mice with Treg cells thatlacked CCR8 displayed a significant reduction in the rate of primarytumor growth (FIG. 9A) as well as lung metastases (FIG. 9B). These dataprovide additional confirmation that, as described herein, CCR8 is animportant means by which Treg cells can modulate anti-tumor immunity.

We also analyzed tumor-infiltrating lymphocytes (TILs) of primary humanbreast carcinomas by flow cytometry. With IRB approval we isolated TILsfrom surgical specimens of patients undergoing surgery for primarybreast cancer. The TILs were analyzed by flow cytometry to determine thephenotypic characteristics of the tumor-infiltrating Treg cells. Thesedata were correlated to tumor grade as determined by the pathologicreview of the tumor specimens. As can be seen, for example, withreference to FIG. 10A, there was a positive correlation between theexpression of CCR8 on Treg cells and their proliferative state (FIG.10A). This finding documents, as described herein, that CCR8 may play arole in the maintenance of Treg cells in the human tumormicroenvironment.

We also compared relative expression of CCR8 on Treg cells infiltratinghuman breast cancer with tumor grade. As shown in FIG. 10B, increasingexpression of CCR8 by tumor infiltrating Treg cells is associated withhigher grade tumors (FIG. 10B).

These data further confirm, as demonstrated herein, that Treg cells areimportant mechanisms of immune tolerance to tumors. Additionally, thesedata further confirm that CCR8 expression by Treg cells contributes totheir ability to thwart anti-tumor immune responses and represents apromising means by which human tumor infiltrating Treg cells can betargeted for the immunotherapy of cancer patients.

REFERENCES

-   1. Dunn G P, Old L J, Schreiber R D. The immunobiology of cancer    immunosurveillance and immunoediting. Immunity 2004; 21(2):137-48.-   2. Hodi F S, O'Day S J, McDermott D F, et al. Improved survival with    ipilimumab in patients with metastatic melanoma. N Engl J Med 2010;    363(8):711-23.-   3. Robert C, Thomas L, Bondarenko I, et al. Ipilimumab plus    dacarbazine for previously untreated metastatic melanoma. N Engl J    Med 2011; 364(26):2517-26.-   4. Galluzzi L, Vacchelli E, Eggermont A, et al. Trial Watch:    Adoptive cell transfer immunotherapy. OncoImmunology 2012;    1(3):306-15.-   5. Kantoff P W, Higano C S, Shore N D, et al. Sipuleucel-T    immunotherapy for castration-resistant prostate cancer. N Engl J Med    2010; 363(5):411-22.-   6. Josefowicz S Z, Niec R E, Kim H Y, et al. Extrathymically    generated regulatory T cells control mucosal TH2 inflammation.    Nature 2012; 482(7385):395-9.-   7. Littman D R, Rudensky A Y. Th17 and regulatory T cells in    mediating and restraining inflammation. Cell 2010; 140(6):845-58.-   8. Samstein R M, Josefowicz S Z, Arvey A, et al. Extrathymic    generation of regulatory T cells in placental mammals mitigates    maternal-fetal conflict. Cell 2012; 150(1):29-38.-   9. Bos P D, Plitas G, Rudra D, et al. Transient regulatory T cell    ablation deters oncogene-driven breast cancer and enhances    radiotherapy. J Exp Med 2013; 210(11):2435-66.-   10. Savage P A, Malchow S, Leventhal D S. Basic principles of    tumor-associated regulatory T cell biology. Trends Immunol 2012.-   11. Galon J, Costes A, Sanchez-Cabo F, et al. Type, density, and    location of immune cells within human colorectal tumors predict    clinical outcome. Science 2006; 313(5795):1960-4.-   12. Sato E, Olson S H, Ahn J, et al. Intraepithelial CD8+    tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell    ratio are associated with favorable prognosis in ovarian cancer.    Proc Natl Acad Sci USA 2005; 102(51):18538-43.-   13. Curiel T J, Coukos G, Zou L, et al. Specific recruitment of    regulatory T cells in ovarian carcinoma fosters immune privilege and    predicts reduced survival. Nat Med 2004; 10(9):942-9.-   14. Zou W. Regulatory T cells, tumour immunity and immunotherapy.    Nat Rev Immunol 2006; 6(4):295-307.-   15. Zheng Y, Chaudhry A, Kas A, et al. Regulatory T-cell suppressor    program co-opts transcription factor IRF4 to control T(H)2    responses. Nature 2009; 458(7236):351-6.-   16. Koch M A, Tucker-Heard G, Perdue N R, et al. The transcription    factor T-bet controls regulatory T cell homeostasis and function    during type 1 inflammation. Nat Immunol 2009; 10(6):595-602.-   17. Chaudhry A, Rudra D, Treuting P, et al. CD4+ regulatory T cells    control TH17 responses in a Stat3-dependent manner. Science 2009;    326(5955):986-91.-   18. Chaudhry A, Rudensky A Y. Control of inflammation by integration    of environmental cues by regulatory T cells. J Clin Invest 2013;    123(3):939-44.-   19. Josefowicz S Z, Lu L F, Rudensky A Y. Regulatory T cells:    mechanisms of differentiation and function. Annu Rev Immunol 2012;    30:531-64.-   20. Kim J M, Rasmussen J P, Rudensky A Y. Regulatory T cells prevent    catastrophic autoimmunity throughout the lifespan of mice. Nat    Immunol 2007; 8(2):191-7.-   21. Pardoll D M. The blockade of immune checkpoints in cancer    immunotherapy. Nat Rev Cancer 2012; 12(4):252-64.-   22. Viguier, M., Lemaitre, F., Verola, O., Cho, M., Gorochov, G.,    Dubertret, L., Bachelez, H., Kourilsky, P., Ferradini, L. (2004).    Foxp3 expressing CD4+CD25high regulatory T cells are overrepresented    in human metastatic melanoma lymph nodes an inhibit the function of    infiltrating T cells. Journal of Immunology, 173: 1444-1453.-   23. Bates, G. J., Fox, S. B., Han, C., Leek, R. D., Garcia, J. F.,    Harris, A. L., Banham, A. H. (2006). Quantification of regulatory T    cells enables the identification of high-risk breast cancer patients    and those at risk of late relapse. Journal of Clinical Oncology,    24(34): 5373-5380.-   24. Mizukami, Y., Kono, K., Kawaguchi, Y., Akaike, H., Kamimura, K.,    Sugai, H., Fujii, H. (2008). Localisation pattern of Foxp3+    regulatory T cells is associated with clinical behaviour in gastric    cancer. British Journal of Cancer, 98(1): 148-153.-   25. Bohling, S. D., Allison, K. H. (2008). Immunosuppressive    regulatory T cells are associated with aggressive breast cancer    phenotypes: a potential therapeutic target. Modern Pathology, 21:    1527-1532.-   26. Wolf, D., Wolf, A. M., Rumpold, H., Fiegl, H., Zeimet, A. G.,    Muller-Holzner, E., Deibl, M., Gastl, G., Gunsilius, E., Marth, C.    (2005). The expression of the regulatory T cell-specific forkhead    box transcription factor foxp3 is associated with poor prognosis in    ovarian cancer. Clinical Cancer Research, 11: 8326-8331.-   27. Hiraoka, N., Onozato, K., Kosuge, T., Hirohashi, S. (2006).    Prevalence of Foxp3+ regulatory T cells increases during the    progression of pancreatic ductal adenocarcinoma and its premalignant    lesions. Clinical Cancer Research, 12(18): 5423-5434.-   28. Pages, F., Galon, J., Dieu-Nosjean, M. C., Tartour, E.,    Sautes-Fridman, C., Fridman, W-H. (2010). Immune infiltration in    human tumors: a prognostic factor that should not be ignored.    Oncogene, 29: 1093-1102.-   29. Loi, S., Sirtaine, N., Piette, F., Salgado, R., Viale, G., Van    Eenoo, F., Rouas, G., Francis, P., Crown, J. P. A., Hitre, E.,    deAzambuja, E., Quinaux, E., Di Leo, A., Michiels, S., Piccart, M.    J., Sotiriou, C. (2013). Prognostic and Predictive Value of    Tumor-Infiltrating Lymphocytes in a Phase III Randomized Adjuvant    Breast Cancer Trial in Node-Positive Breast Cancer Comparing the    Addition of Docetaxel to Doxorubicin With Doxorubicin-Based    Chemotherapy: BIG 02-98. Journal of Clinical Oncology, 31(5):    860-867.-   30. Prall, F., Duhrkop, T., Weirich, V., Ostwald, C., Lenz, P.,    Nizze, H., Barten, M. (2004). Prognostic role of CD8+    tumor-infiltrating lymphocytes in stage III colorectal cancer with    and without microsatellite instability. Human Pathology, 35(7):    808-816.-   31. Sharma, P., Wagner, K., Wolchok, J. D., Allison, J. P. (2011).    Novel cancer immunotherapy agents with survival benefit: recent    successes and next steps. Nature Review Cancer, 11: 805-812.-   32. Ott, P. A., Hodi, F. S., Robert, C. (2013). CTLA-4 and    PD-1/PD-L1 blockade: new immunotherapeutic modalities with durable    clinical benefit in melanoma patients. Clinical Cancer Resolution,    19: 5300-5309.-   33. Simpson, T. R., Li, F., Montalvo-Ortiz, W., Sepulveda, M. A.,    Bergerhoff, K., Arce, F., Roddie, C., Henry, J. Y., Yagita, H.,    Wolchok, J. D., Peggs, K. S., Ravetch, J. V., Allison, J. P.,    Quezada, S. A. (2013). Fc-dependent depletion of tumor-infiltrating    regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy    against melanoma. Journal of Experimental Medicine,    210(9):1695-1710.-   34. Leach, D. R., Krummel, M. F., Allison, J. P. (1996). Enhancement    of antitumor immunity by CTLA-4 blockade. Science, 271(5256):    1734-1736.-   35. Scott, A. M., Wolchok, J. D., Old, L. J. (2012). Antibody    therapy of cancer. Nature Reviews Cancer, 12: 278-287.-   36. Fontenot, J. D., Gavin, M. A., Rudensky, A. Y. (2003). Foxp3    programs the development and function of CD4+CD25+ regulatory T    cells. Nature, 4(4): 330-336.-   37. Yagi, H., Nomura, T., Nakamura, K., Yamazaki, S., Kitawaki, T.,    Hori, S., Maeda, M., Onodera, M, Uchiyama, T., Fujii, S.,    Sakaguchi, S. (2004). Crucial role of FOXP3 in the development and    function of human CD25+CD4+ regulatory T cells. International    Immunology, 16(11): 1643-1656.-   38. Klages, K., Mayer, C. T., Lahl, K., Loddenkemper, C., Teng, M.    W., Ngiow, M. W., Smyth, M. J., Hamann, A., Huehn, J.,    Sparwasser, T. (2010). Selective depletion of Foxp3+ regulatory T    cells improves therapeutic vaccination against established melanoma.    Cancer Research, 70(20): 7788-7799.-   39. Teng, M. W., Ngiow, S. F., von Scheidt, B., McLaughlin, N.,    Sparwasser, T., Smyth, M. J. (2010). Conditional regulatory T-cell    depletion releases adaptive immunity preventing carcinogenesis and    suppressing established tumor growth. Cancer Research,    70(20):7800-7809.-   40. Kim, J. M., Lahl, K., Hori, S., Loddenkemper, C., Chaudhry, A.,    deRoos, P., Rudensky, A., Sparwasser, T. (2009). Cutting edge:    depletion of Foxp3+ cells leads to induction of autoimmunity by    specific ablation of regulatory T cells in genetically targeted    mice. Journal of Immunology, 182(12): 7631-7634.-   41. Chinen, T., Volchkov, P. Y., Chervonsky, A. V., Rudensky, A. Y.    (2010). A critical role for regulatory T cell-mediated control of    inflammation in the absence of commensal bacteria. Journal of    Experimental Medicine, 207(11): 2323-2330.-   42. Bindea, G., Mlecnik, B., Tosolini, M., Kirilovsky, A., Waldner,    M., Obenauf, A. C., Angell, H., Fredrikson, T., Lafontaine, L.,    Berger, A., Bruneval, P., Fridman, W. H., Becker, C., Pages, F.,    Speicher, M. R., Trajanoski, Z., Galon, J. (2013). Spatiotemporal    dynamics of intratumoral immune cells reveal the immune landscape in    human cancer. Immunity, 39(4): 782-795.-   43. Li, Y., Xie, X. (2013). A mixture model for expression    deconvolution from RNA-seq in heterogeneous tissues. BMC    Bioinformatics, 14(Suppl 5): S11.-   44. Islam, S. A., Ling, M. F., Leung, J., Shreffler, W. G.,    Luster, A. D. (2013). Identification of human CCR8 as a CCL18    receptor. Journal of Experimental Medicine, 210(10): 1889-1898.-   45. Facciabene, A., Peng, X., Hagemann, I. S., Balint, K.,    Barchetti, A., Wang, L. P., Gimotty, P. A., Gilks, C. B., Lal, P.,    Zhang, L., Coukos, G. (2011). Tumor hypoxia promotes tolerance and    angiogenesis via CCL28 and Treg cells. Nature, 475(7355): 226-230.-   46. Hori, S., Nomura, T., Sakaguchi, S. (2003). Control of    regulatory T cell development by the transcription factor Foxp3.    Science, 299(5609): 1057-1061.-   47. Bennett, C. L., Christie, J., Ramsdell, F., Brunkow, M. E.,    Ferguson, P. J., Whitesell, L., Kelly, T. E., Saulsbury, F. T.,    Chance, P. F., Ochs, H. D. (2001). The immune dysregulation,    polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused    by mutations of FOXP3. Nature Genetics, 27(1):20-21.-   48. Williams, L. M. and Rudensky, A. Y. (2007). Maintenance of the    Foxp3-dependent developmental program in mature regulatory T cells    requires continued expression of Foxp3. Nature Immunology, 8(3):    277-284.-   49. Homo sapiens ATCC® CRL-8294™-   50. Shalem, O., Sanjana, N. E., Hartenian, E., Shi, X., Scott, D.    A., Mikkelsen, T. S., Heckl, D., Ebert, B. L., Root, D. E.,    Doench, J. G., Zhang, F. (2014). Genome-scale CRISPR-Cas9 knockout    screening in human cells. Science, 343(6166): 84-87.-   51. Gavin, M. A., Rasmussen, J. P., Fontenot, J. D., Vasta, V.,    Manganiello, V. C., Beavo, J. A., Rudensky, A. Y. (2007).    Foxp3-dependent programme of regulatory T-cell differentiation.    Nature, 445: 771-775.-   52. Yang, H., Wang, H., Shivalila, C. S., Cheng, A. W., Shi, L.,    Jaenisch, R. (2013). One-step generation of mice carrying reporter    and conditional alleles by CRISPR/Cas-mediated genome editing. Cell,    154: 1370-1379.-   53. Lal, G., Zhang, N., van der Touw, W., Ding, Y., Ju, W.,    Bottinger, E. P., Reid, S. P., Levy, D. E., Bromberg, J. S. (2009).    Epigenetic regulation of Foxp3 expression in regulatory T cells by    DNA methylation. Journal of Immunology, 182(1): 259-273.-   54. Polansky, J. K., Kretschmer, K., Freyer, J., Floess, S., Garbe,    A., Baron, U., Olek, S., Hamann, A., von Boehmer, H., Huehn, J.    (2008). DNA methylation controls Foxp3 gene expression. European    Journal of Immunology, 38: 1654-1663.-   55. Wang, L., Liu, Y., Beier, U. H., Han, R., Bhatti, T. R.,    Akimova, T., Hancock, W. W. (2013). Foxp3+ T-regulatory cells    require DNA methyltransferase 1 expression to prevent development of    lethal autoimmunity. Blood, 121: 3631-3639.-   56. Schmidt, A. M., Zou, T., Joshi, R. P., Leichner, T. M.,    Pimentel, M. A., Sommers, C. L., Kambayashi, T. (2013).    Diacylglycerol kinase z limits the generation of natural regulatory    T cells. Science Signaling, 6(303): 101-111.-   57. Buckner, J. H. (2010). Mechanisms of impaired regulation by    CD4+CD25+FOXP+ regulatory T cells in human autoimmune diseases.    Nature Reviews Immunology, 10: 849-859.-   58. Fellman, C., Hoffman, T., Sridhar, V., Hopfgartner, B., Muhar,    M., Roth, M., Lai, D. Y., Barbosa, I. A. M., Kwon, J. S., Guan, Y.,    Sinha, N., Zuber, J. (2013). An optimized microRNA backbone for    effective single-copy RNAi. Cell Reports, 5(6): 1704-1713.-   59. Mulligan A M, Raitman I, Feeley L, et al. Tumoral lymphocytic    infiltration and expression of the chemokine CXCL10 in breast    cancers from the Ontario Familial Breast Cancer Registry. Clinical    cancer research: an official journal of the American Association for    Cancer Research 2013; 19:336-46-   60. Yan M, Jene N, Byrne D, et al. Recruitment of regulatory T cells    is correlated with hypoxia-induced CXCR4 expression, and is    associated with poor prognosis in basal-like breast cancers. Breast    cancer research: BCR 2011; 13:R47-   61. Deleeuw, et al. Clin. Cancer Res. 18, 3022, 2012)-   62. T. Chinen, A. Y. Rudensky, The effects of commensal microbiota    on immune cell subsets and inflammatory responses. Immunological    reviews 245, 45 (January 2012).-   63. D. G. DeNardo et al., CD4(+) T cells regulate pulmonary    metastasis of mammary carcinomas by enhancing protumor properties of    macrophages. Cancer cell 16, 91 (Aug. 4, 2009).-   64. L. V. Hooper, D. R. Littman, A. J. Macpherson, Interactions    between the microbiota and the immune system. Science 336, 1268    (Jun. 8, 2012).-   65. J. W. Verbsky, T. A. Chatila, Immune dysregulation,    polyendocrinopathy, enteropathy, X-linked (IPEX) and IPEX-related    disorders: an evolving web of heritable autoimmune diseases. Current    opinion in pediatrics 25, 708 (December 2013).-   66. Y. P. Rubtsov et al., Regulatory T cell-derived interleukin-10    limits inflammation at environmental interfaces. Immunity 28, 546    (April 2008).-   67. M. Y. Gerner, W. Kastenmuller, I. Ifrim, J. Kabat, R. N.    Germain, Histo-cytometry: a method for highly multiplex quantitative    tissue imaging analysis applied to dendritic cell subset    microanatomy in lymph nodes. Immunity 37, 364 (Aug. 24, 2012).-   68. F. Barzaghi, L. Passerini, R. Bacchetta, Immune dysregulation,    polyendocrinopathy, enteropathy, x-linked syndrome: a paradigm of    immunodeficiency with autoimmunity. Frontiers in immunology 3, 211    (2012).-   69. T. Ishida et al., Stevens-Johnson Syndrome associated with    mogamulizumab treatment of adult T-cell leukemia/lymphoma. Cancer    Sci 104, 647 (May, 2013).-   70. H. Azukizawa, S. Sano, H. Kosaka, Y. Sumikawa, S. Itami,    Prevention of toxic epidermal necrolysis by regulatory T cells.    European Journal of Immunology 35, 1722 (June 2005).-   71. R. Takahashi et al., Defective regulatory T cells in patients    with severe drug eruptions: timing of the dysfunction is associated    with the pathological phenotype and outcome. J Immunol 182, 8071    (Jun. 15, 2009).-   72. E. Antiga et al., Characterization of regulatory T cells in    patients with dermatomyositis. Journal of autoimmunity 35, 342    (December 2010).-   73. Comprehensive molecular portraits of human breast tumours.    Nature 490, 61 (Oct. 4, 2012).-   74. C. M. Perou, Molecular stratification of triple-negative breast    cancers. The oncologist 16 Suppl 1, 61 (2011).-   75. E. A. Rakha, J. S. Reis-Filho, I. O. Ellis, Basal-like breast    cancer: a critical review. Journal of clinical oncology: official    journal of the American Society of Clinical Oncology 26, 2568 (May    20, 2008).-   76. A. M. Mulligan et al., Tumoral lymphocytic infiltration and    expression of the chemokine CXCL10 in breast cancers from the    Ontario Familial Breast Cancer Registry. Clinical cancer research:    an official journal of the American Association for Cancer Research    19, 336 (Jan. 15, 2013).-   77. M. Yan et al., Recruitment of regulatory T cells is correlated    with hypoxia-induced CXCR4 expression, and is associated with poor    prognosis in basal-like breast cancers. Breast cancer research: BCR    13, R47 (2011).-   78. D. M. Parkin, Global cancer statistics in the year 2000. The    lancet oncology 2, 533 (September 2001).-   79. P. C. Enzinger, R. J. Mayer, Esophageal cancer. The New England    journal of medicine 349, 2241 (Dec. 4, 2003).-   80. A. Amedei, M. Benagiano, C. della Bella, E. Niccolai, M. M.    D'Elios, Novel immunotherapeutic strategies of gastric cancer    treatment. Journal of biomedicine & biotechnology 2011, 437348    (2011).-   81. L. Shi et al., Efficacy of adjuvant immunotherapy with    cytokine-induced killer cells in patients with locally advanced    gastric cancer. Cancer immunology, immunotherapy: CII 61, 2251    (December 2012).-   82. S. Zhou et al., CCR7 Expression and Intratumoral FOXP3(+)    Regulatory T Cells are Correlated with Overall Survival and Lymph    Node Metastasis in Gastric Cancer. PloS one 8, e74430 (2013).-   83. T. Sasada, M. Kimura, Y. Yoshida, M. Kanai, A. Takabayashi,    CD4+CD25+ regulatory T cells in patients with gastrointestinal    malignancies: possible involvement of regulatory T cells in disease    progression. Cancer 98, 1089 (Sep. 1, 2003).-   84. B. Deng et al., Intratumor hypoxia promotes immune tolerance by    inducing regulatory T cells via TGF-beta1 in gastric cancer. PloS    one 8, e63777 (2013).-   85. K. Cetin, D. S. Ettinger, Y. J. Hei, C. D. O'Malley, Survival by    histologic subtype in stage IV nonsmall cell lung cancer based on    data from the Surveillance, Epidemiology and End Results Program.    Clinical epidemiology 3, 139 (2011).-   86. L. V. Kalialis, K. T. Drzewiecki, H. Klyver, Spontaneous    regression of metastases from melanoma: review of the literature.    Melanoma research 19, 275 (October 2009).-   87. A. Shablak et al., High-dose interleukin-2 can produce a high    rate of response and durable remissions in appropriately selected    patients with metastatic renal cancer. J Immunother 34, 107 (January    2011).-   88. K. I. Al-Shibli et al., Prognostic effect of epithelial and    stromal lymphocyte infiltration in non-small cell lung cancer.    Clinical cancer research: an official journal of the American    Association for Cancer Research 14, 5220 (Aug. 15, 2008).-   89. K. Shimizu et al., Tumor-infiltrating Foxp3+ regulatory T cells    are correlated with cyclooxygenase-2 expression and are associated    with recurrence in resected non-small cell lung cancer. Journal of    thoracic oncology: official publication of the International    Association for the Study of Lung Cancer 5, 585 (May, 2010).-   90. R. P. Petersen et al., Tumor infiltrating Foxp3+ regulatory    T-cells are associated with recurrence in pathologic stage I NSCLC    patients. Cancer 107, 2866 (Dec. 15, 2006).-   91. K. Suzuki et al., Clinical impact of immune microenvironment in    stage I lung adenocarcinoma: tumor interleukin-12 receptor beta2    (IL-12Rbeta2), IL-7R, and stromal FoxP3/CD3 ratio are independent    predictors of recurrence. Journal of clinical oncology: official    journal of the American Society of Clinical Oncology 31, 490 (Feb.    1, 2013).-   92. Y. Q. He et al., FoxP3 genetic variants and risk of non-small    cell lung cancer in the Chinese Han population. Gene 531, 422 (Dec.    1, 2013).-   93. J. R. Brahmer et al., Safety and activity of anti-PD-L1 antibody    in patients with advanced cancer. The New England journal of    medicine 366, 2455 (Jun. 28, 2012).-   94. S. L. Topalian et al., Safety, activity, and immune correlates    of anti-PD-1 antibody in cancer. The New England journal of medicine    366, 2443 (Jun. 28, 2012).-   95. Nat Immunol. 2011 February; 12(2):167-77. doi: 10.1038/ni.1984.    Epub 2011 Jan. 9.-   96. Biochem Pharmacol. 2012 Mar. 15; 83(6):778-87. doi:    10.1016/j.bcp.2011.12.021. Epub 2011 Dec. 24.-   97. Adv Immunol. 2006; 90:83-131.-   98. Deleeuw, et al. Clin. Cancer Res. 18, 3022, 2012

1.-9. (canceled)
 10. A method of detecting and/or characterizing a tumorby administering to a subject having the tumor a composition comprisinga CCR8-targeting agent in association with a detectable moiety, so thattumor-infiltrating Treg cells are detected.
 11. The method of claim 10,further comprising treating the subject in which tumor-infiltrating Tregcells are detected by targeting CCR8.
 12. The method of claim 10,further comprising treating the subject in which tumor-infiltrating Tregcells are detected by targeting CCR4.
 13. The method of claim 12,wherein targeting CCR4 comprises administering to the subject acomposition comprising a CCR4-targeting agent.
 14. The method of claim13, wherein the CCR4-targeting agent is or comprises a bifunctionalagent that targets both CCR8 and CCR4.
 15. The method of claim 10,further comprising administering one or more immunomodulatorytherapeutic modalities to the subject. 16.-24. (canceled)
 25. The methodof claim 10, wherein the detectable moiety is a radioactive,fluorescent, chemiluminescent, or phosphorescent moiety.